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)

IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 microM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta-treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.
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PMID:Effect of IL-1beta on CRE-dependent gene expression in human airway smooth muscle cells. 1238 41

To investigate regulation of D2 receptor (D2R) gene expression by protein kinases, we evaluated effects of constitutively active MAPK kinase kinase (MEKK), Ca2+/calmodulin-dependent protein kinase (CaMK) II, CaMKIV and cyclic AMP-dependent protein kinase (PKA) on D2R promoter activity using luciferase reporter gene assays. A 1.5-kbp fragment containing the rat D2R promoter was cloned upstream of the reporter and transfected into D2R-expressing NB2A cells or nonexpressing NG108-15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV and PKA, increased promoter activity 4.5- and 1.5-fold, respectively, in NB2A cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant negative (DN)-JNK1 or DN-p38MAPK revealed that ERK but not JNK and p38MAPK is involved in MEKK-induced promoter activation. Deletion and mutation of the promoter revealed that the MEKK-responsive region was Sp1 site B between nucleotides -56 and -47. Overexpression of Sp1 suppressed promoter activity without affecting MEKK-induced activation. Interestingly, overexpression of Zif268 increased promoter activity through the region between nucleotides -56 and -36. Increased activity by Zif268 was additive with CaMKII-induced activation but not with activation induced by MEKK. Co-transfection with CaMKII stimulated nuclear translocation of Zif268. These results suggest that ERK and CaMKII positively regulate the D2R promoter and that Zif268 is a potential transcription factor for the CaMKII-dependent pathway.
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PMID:Activation of the rat dopamine D2 receptor promoter by mitogen-activated protein kinase and Ca2+/calmodulin-dependent protein kinase II pathways. 1242 50

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a 32-kDa serine protease inhibitor that is associated with the extracellular matrix. hTFPI-2 inhibits several extracellular matrix-degrading serine proteases and may play a role in tumor invasion and metastasis. To study the signal transduction pathway that leads to the activation of the hTFPI-2, we cloned the potential promoter region of this gene adjacent to a heterologous luciferase reporter gene. Phorbol 12-myristate 13-acetate (PMA) induced the luciferase reporter gene in HEK293 cells and other epithelial cell lines, such as the human lung carcinoma A549 cells, the breast carcinoma MCF7 cells, and the cervical HeLa cells. This PMA induction was blocked with the MEK inhibitor UO126, suggesting that the PMA-induced activation of the hTFPI-2 promoter is mediated through MEK. Furthermore, epidermal growth factor induced the luciferase reporter gene in HeLa cells. Cotransfection of the luciferase construct with constitutively active components of the Ras/Raf/MEK/ERK pathway in EcR-293 cells lead to a 7- to 92-fold induction of the luciferase reporter gene, indicating that regulation of hTFPI-2 is mediated through this pathway. A series of luciferase reporter gene constructs with progressive deletions of the 5'-flanking region suggested that the minimal basal promoter activity is located between nucleotide positions -89 and -384, whereas the minimal inducible promoter activity is between -89 and -222. We have used the computer program TFSEARCH and mutagenesis to analyze potential transcription factor binding sites. We identified an AP-1 binding site at nucleotide position -156 (inducible activity) and a Sp1 site at position -134 (basal activity) as potential cis-acting elements in the promoter region of the hTFPI-2.
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PMID:The ERK/MAPK pathway regulates the activity of the human tissue factor pathway inhibitor-2 promoter. 1244 83

Expression of 12 matrix metalloproteinases (MMPs) after exposure of human melanoma cell lines C32TG and Mewo to nitric oxide (NO) was investigated by the reverse transcription-polymerase chain reaction. Expression of the mRNA of MMP-1, -3, -10 and -13 in C32TG cells was transcriptionally enhanced in a dose-dependent manner by exposure to an NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) and mRNA expression of MMP-1 and -10 was similarly enhanced in Mewo cells. Exposure of C32TG cells to NO increased the MMP-1 protein concentration in the culture medium. Testing with the luciferase gene fused to the 1.5 Kbp 5'-flanking region of the human MMP-1 gene showed that exposure to NO upregulated MMP-1 promoter activity in C32TG cells. Endogenous NO production after introduction of inducible NO synthase cDNA also enhanced MMP-1 promoter activity in C32TG cells. Deletion and mutational analysis identified a critical AP-1 binding site required for NO regulation of MMP-1. A neighboring Ets motif from the AP-1 site in the promoter region acted as an accessory to enhance MMP-1 expression. Electromobility shift analysis using the AP-1 binding site showed that NO enhanced the AP-1 binding ability of nuclear factors in C32TG cells. PD98059, a selective MEK inhibitor and SB202190, a p38 MAPK inhibitor, attenuated the MMP-1 mRNA expression enhanced by NO. Thus, MMP-1 was transcriptionally enhanced by NO via MAPK (ERK and p38) pathways. The results of our study suggest that the increased expression of MMPs in response to NO may be associated with tumor progression under inflammation.
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PMID:Induction of matrix metalloproteinase gene transcription by nitric oxide and mechanisms of MMP-1 gene induction in human melanoma cell lines. 1245 29

The purpose of the current study was to determine whether nuclear factor-kappaB (NF-kappaB) activation is a component of the depolarization/Ca(2+)-dependent signaling in beta-cells. MIN6 cells were transfected with a plasmid containing five tandem repeats of NF-kappaB binding sites linked to a luciferase reporter. The results of these experiments showed that KCl induced depolarization-activated NF-kappaB-dependent transcription (3.8-fold at 45 mmol/l, P < 0.01) in a concentration-dependent manner. Tumor necrosis factor-alpha (TNF-alpha), a known inducer of NF-kappaB signaling, activated this construct by 3.4-fold (P < 0.01). The response of NF-kappaB to depolarization was inhibited by the Ca(2+)-channel blocker verapamil and by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (70 and 62%, respectively). TNF-alpha, glucose, and KCl treatment resulted in inhibitory kappaBalpha degradation by Western blot analysis. TNF-alpha treatment and depolarization activation of NF-kappaB differed significantly in that TNF-alpha activation was not blocked by PD98059. Transfection with PKA, MEK, and MEK kinase induced NF-kappaB-dependent transcription by 20-, 90-, and 300-fold, respectively, suggesting that these pathways contribute to the activation in the depolarization response. These findings demonstrate that depolarization/Ca(2+) influx, as well as TNF-alpha treatment, can activate NF-kappaB-dependent transcription in pancreatic beta-cells, but by different signaling pathways. The current studies show that Ca(2+) signals in pancreatic beta-cells can activate transcription factors involved in the regulation of cell cycle and apoptosis. These findings now add NF-kappaB to the list of depolarization-induced transcription factors in pancreatic beta-cells.
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PMID:Activation of nuclear factor-kappaB by depolarization and Ca(2+) influx in MIN6 insulinoma cells. 1247 94

Enhanced VEGF-A (vascular endothelial growth factor A) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies including gastric cancer. Oxidative stress has been linked to tumor-associated neoangiogenesis; underlying mechanisms, however, remained poorly understood. Therefore, we studied the effect of oxidative stress on VEGF-A gene expression in gastric cancer cells. Oxidative stress generated by H(2)O(2) application potently stimulated VEGF-A protein and mRNA levels as determined by enzyme-linked immunosorbent assay and real-time PCR techniques, respectively, and elevated the activity of a transfected (-2018) VEGF-A promoter reporter gene construct in a time- and dose-dependent manner (4-8-fold). These effects were abolished by the antioxidant N-acetylcysteine, demonstrating specificity of oxidative stress responses. Functional 5' deletion analysis mapped the oxidative stress response element of the human VEGF-A promoter to the sequence -88/-50, and a single copy of this element was sufficient to confer basal promoter activity as well as oxidative stress responsiveness to a heterologous promoter system. Combination of EMSA studies, Sp1/Sp3 overexpression experiments in Drosophila SL-2 cells, and systematic promoter mutagenesis identified enhanced Sp1 and Sp3 binding to two GC-boxes at -73/-66 and -58/-52 as the core mechanism of oxidative stress-triggered VEGF-A transactivation. Additionally, in Gal4-Sp1/-Sp3-Gal4-luciferase assays, oxidative stress increased Sp1 but not Sp3 transactivating capacity, indicating additional mechanism(s) of VEGF-A gene regulation. Signaling studies identified a cascade comprising Ras --> Raf --> MEK1 --> ERK1/2 as the main pathway mediating oxidative stress-stimulated VEGF-A transcription. This study for the first time delineates the mechanisms underlying regulation of VEGF-A gene transcription by oxidative stress and thereby further elucidates potential pathways underlying redox control of neoangiogenesis.
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PMID:Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements. 1250 26

Persistent proteinuria has been indicated to be a major risk factor for the development of tubulointerstitial damage through a process of proinflammatory molecule expression. Monocyte chemoattractant protein-1 (MCP-1) was shown to contribute to recruitment of immune cells into the renal interstitium in acute and chronic renal diseases. However, the molecular mechanisms by which proteinuria causes MCP-1 expression in proximal tubular cells have not been fully clarified. In this study, we examined whether albumin overload-induced MCP-1 expression was regulated by mitogen-activated protein kinase (MAPK) in mouse proximal tubular (mProx) cells. Exposure of mProx cells to delipidated bovine serum albumin (BSA) induced mRNA and protein expression of MCP-1 in a time- and dose-dependent manner. BSA activated extracellular signal-regulated kinase (ERK1/2) and p38 MAPK. The MEK inhibitor U-0126 partially suppressed BSA-induced MCP-1 expression and MCP-1 promoter/luciferase reporter activity. U-0126 also inhibited an increase in nuclear factor-kappaB and activator protein-1 DNA-binding activity of MCP-1 promoter by protein overload in mProx cells. In addition, we found that U-0126 inhibited BSA-induced nuclear factor-kappaB reporter activity and inhibitory protein degradation in mProx cells. In conclusion, these findings indicate that ERK signaling is involved in BSA-induced MCP-1 expression in mProx cells.
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PMID:Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells. 1251 35

Activation of G-protein coupled muscarinic acetylcholine receptors and MAPKs/ERK-1/2 has been found to inhibit neural cell apoptosis and promote neural cell survival. Bcl-2 protein family also plays an important role in regulating neural cell apoptosis and survival. However, signaling pathways coupling muscarinic receptors to Bcl-2 family remains to be elucidated. In the present study, it was found that carbachol not only activated MEK/ERK-1/2 signaling pathways, but also increased the expression levels of Bcl-2 and phospho-Bad proteins in human neuroblastoma SH-SY5Y cells. These effects were blocked by a muscarinic receptor antagonist (atropine) and a MEK inhibitor(PD98059) and were significantly attenuated by a Src family kinases inhibitor(PP1) and a PKC inhibitor (bisindolymaleimide-I), but were not influenced by a G(i/o)-uncoupling reagent (pertussin toxin) and a PI-3 kinase inhibitor (wortmannin). Furthermore, carbachol also stimulated Bcl-2 promoter-driven luciferase gene expression in transfected SH-SY5Y cells. Co-transfection of Ras or Raf dominant negative mutants with the pBcl-2-Luc plasmid abolished carbachol s effects. These data suggested that muscarinic acetylcholine receptors regulated the expression of Bcl-2 protein family by Ras-ERK-1/2 signaling pathway involving the pertussin toxin-insensitive G-proteins, PKC and Src.
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PMID:[G-protein-coupled muscarinic acetylcholine receptor activation up-regulates Bcl-2 and phospho-bad via Ras-ERK-1/2 signaling pathway]. 1251 26

We have recently demonstrated that relaxin (RLX) acts as compensatory mediator in human heart failure. RLX inhibits the stimulation of endothelin-1, the most potent vasoconstrictor in heart failure. Upregulation of the endothelin type-B receptor (ET(B)), which mediates endothelin-1 clearance and endothelial release of NO, represents a pivotal mode of RLX action. However, signal transduction and abundance of this phenomenon are unknown. Therefore, we investigated RLX-induced regulation of ET(B) in human umbilical vein endothelial, epithelial (HeLa), and vascular smooth muscle cells. In human umbilical vein endothelial cells and HeLa cells, but not in human vascular smooth muscle cells, RLX upregulated ET(B) expression and activated extracellular signal-regulated kinase-1/2 (ERK-1/2) and nuclear factor-kappaB (NF-kappaB), a transcription factor. PD-98059, a selective inhibitor of the mitogen-activated protein kinase kinase-1 (MEK-1)-ERK-1/2 pathway, abolished ERK-1/2 and NF-kappaB activation and ET(B) upregulation. NF-kappaB inhibition also prevented RLX-mediated ET(B) stimulation. In NF-kappaB-luciferase reporter assays, we demonstrated complete inhibition of RLX-induced NF-kappaB activation in cells transfected with dominant-negative Raf-1, MEK-1, or ERK-1/2 constructs, whereas dominant-negative Ras had no effect. In rat aorta and mesenteric artery, RLX pretreatment, in an ET(B)-dependent fashion, mitigated the maximum contractile response to endothelin-1, by 38+/-4% and 43+/-6%, and the endothelin-1 sensitivity (-log[EC(50)]: aorta, 8.2+/-0.2 for vehicle versus 7.2+/-0.2 for RLX; mesenteric artery, 8.0+/-0.2 for vehicle versus 7.1+/-0.1 for RLX). RLX pretreatment augmented the dilator effect of the ET(B) agonist endothelin-3 by 100+/-8% and 133+/-13%. In conclusion, RLX stimulates endothelial and epithelial ET(B) via a Ras-independent Raf-1-MEK-1-ERK-1/2 pathway that activates NF-kappaB. On vascular smooth muscle cells, ET(B), a contributor to endothelin-mediated vasoconstriction, remains unaffected. This renders RLX a functional endothelin-1 antagonist.
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PMID:Relaxin, a pregnancy hormone, is a functional endothelin-1 antagonist: attenuation of endothelin-1-mediated vasoconstriction by stimulation of endothelin type-B receptor expression via ERK-1/2 and nuclear factor-kappaB. 1252 18

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disease, the cause of which is unknown. Diagnostic of HCL is abnormal expression of the gene that encodes the beta2 integrin CD11c. In order to determine the cause of CD11c gene expression in HCL the CD11c gene promoter was characterized. Transfection of the CD11c promoter linked to a luciferase reporter gene indicated that it is sufficient to direct expression in hairy cells. Mutation analysis demonstrated that of predominant importance to the activity of the CD11c promoter is its interaction with the activator protein-1 (AP-1) family of transcription factors. Comparison of nuclear extracts prepared from hairy cells with those prepared from other cell types indicated that hairy cells exhibit abnormal constitutive expression of an AP-1 complex containing JunD. Functional inhibition of AP-1 expressed by hairy cells reduced CD11c promoter activity by 80%. Inhibition of Ras, which represents an upstream activator of AP-1, also significantly inhibited the CD11c promoter. Furthermore, in the hairy cell line EH, inhibition of Ras signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1 and 2 (MEK1/2) reduced not only CD11c promoter activity but also reduced both CD11c surface expression and proliferation. Expression in nonhairy cells of a dominant-positive Ras mutant activated the CD11c promoter to levels equivalent to those in hairy cells. Together, these data indicate that the abnormal expression of the CD11c gene characteristic of HCL is dependent upon activation of the proto-oncogenes ras and junD.
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PMID:CD11c gene expression in hairy cell leukemia is dependent upon activation of the proto-oncogenes ras and junD. 1257 24


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