Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Agonist activity at G protein-coupled receptors (GPCRs) that regulate heterotrimeric G proteins of the Galpha(i/o) or Galpha(q) families has been shown to result in activation of the mitogen-activated protein (MAP) kinase cascade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary complex transcription factor Sap1a following MAP kinase activation. In contrast to other reporter gene assays for Galpha(i/o)-coupled GPCRs, the MAP kinase reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to generate a "host" reporter gene-containing cell line. The Galpha(i)-coupled human CXCR1 chemokine receptor was subsequently transfected into this cell line in order to develop a 384-well format screen for both agonists and antagonists of this receptor. Agonists activated the reporter gene with the expected rank order of potency and with similar concentration dependence as seen with the regulation of other signal transduction cascades in mammalian cells: interleukin-8 (IL-8) (pEC(50) = 7.0 +/- 0.1) > GCP-2 (pEC(50) = 6.3 +/- 0.1) > NAP-2 (pEC(50) < 6). CXCR1-mediated activation of MAP kinase was inhibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating that receptor activation of MAP kinase is due to pertussis toxin-sensitive Galpha(i/o)-family G proteins to cause the activation of MEK kinase. Using the 384-well format, assay performance was unaffected by solvent concentrations of 0.5% ethanol, 0.15% glycerol, or 1% DMSO. Signal crosstalk between adjacent wells was less than 1%. The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.
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PMID:Development of a homogeneous MAP kinase reporter gene screen for the identification of agonists and antagonists at the CXCR1 chemokine receptor. 1167 62

We used cytokine protein array to analyze the expression of cytokines from human cord blood-derived mesenchymal stem cells (CB-MSCs). Several cytokines, interleukins (IL), and growth factors, including ENA-78, GM-CSF, GRO, IL-1beta, IL-6, IL-8, MCP-1, OSM, VEGF, FGF-4, FGF-7, FGF-9, GCP-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IP-10, LIF, MIF, MIP-3alpha, osteoprotegerin, PARC, PIGF, TGF-beta2, TGF-beta3, TIMP-1, as well as TIMP-2, were secreted by CB-MSCs, while IL-4, IL-5, IL-7, IL-13, TGF-beta1, TNF-alpha, and TNF-beta were not expressed under normal growth conditions. IL-6, IL-8, TIMP-1, and TIMP-2 were the most abundant interleukins expressed by CB-MSCs. A set of growth factors were selected to evaluate their stimulatory effects on the IL6 secretion for CB-MSCs. IL-1beta was the most important factor inducing CB-MSC to secret IL-6. The mechanism by which IL-1beta promoted IL-6 expression in CB-MSCs was studied. By using various inhibitors of signal transduction, we found that activation of p38 mitogen-activated protein kinases (MAPK) and MAPK kinase (MEK) is essential in the IL-1beta stimulated signaling cascade which leads to the increase in IL-6 synthesis. Additionally, continuous supplement of IL-1beta in the CB-MSCs culture will facilitate adipogenic maturation of CB-MSCs as evidenced by the presence of oil drops in the CB-MSCs and secretion of leptin, a molecule marker of adipocytes. These results strongly suggest that cytokine induction and signal transduction are important for the differentiation of CB-MSCs.
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PMID:Cytokine interactions in mesenchymal stem cells from cord blood. 1637 3