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)

Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. However, the underlying molecular mechanisms remain obscure. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-12) M) EGF concentrations in A431 cells. The time course of AR messenger RNA (mRNA) accumulation was different with 10(-12) and 10(-9) M EGF; AR induction by 10(-9) M EGF peaked between 1 and 1.5 h, then decreased to the basal level within 2 h. Conversely, the induction by 10(-12) M EGF was slightly delayed, but persisted for 4 h. The involvement of tyrosine phosphorylation in AR induction by EGF was suggested by the ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) M EGF. In the presence of the protein phosphatase 2A inhibitor, okadaic acid, 10(-9) M EGF induced a persistent accumulation of AR mRNA. On the contrary, okadaic acid abrogated the stimulation of AR mRNA level induced by a low EGF concentration, suggesting that both EGF concentrations activated distinct regulatory mechanisms. The signaling components involved in the differential activities of EGF in A431 cells were then examined. We previously reported a relationship between the ambivalent activity of EGF and the p42-mitogen-activated protein (MAP) kinase activity. Thus, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 10(-9) M EGF led to a sharp, but transitory, activation. The MAP kinases are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no significant effect of 10(-12) M EGF could be detected, 10(-9) M EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction by 10(-9) M, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the involvement of c-Raf-1 in the signaling pathway induced by EGF was verified. A concentration of 10(-9) M EGF induced stimulation of c-Raf-1 kinase activity, whereas 10(-12) M EGF not only failed to activate c-Raf-1, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differently affect gene expression and cell proliferation through distinct mechanisms of regulation.
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PMID:Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a signal transduction pathway that can bypass Raf-1 activation. 956 49

Epithelial cell differentiation is regulated by specific combinations of growth factors, hormones, and extracellular matrix (ECM). How these divergent signals are integrated is largely unknown. We used primary cultures of normal human bronchial epithelial cells (NHBEs) to investigate mechanisms of signal integration. In defined, serum-free media, NHBEs undergo mucosecretory differentiation only when grown in the presence of retinoids and on the appropriate substratum (collagen gels). We identified the retinoic acid receptor beta (RARbeta) gene as an early marker of NHBE differentiation. In contrast to immortalized cell lines, in NHBEs strong retinoid-induced RARbeta transcription occurs only when cells are grown on collagen gels, and it requires new protein synthesis and a cis-acting element that maps outside the known RARbeta promoter elements. NHBEs grown on collagen gels exhibit reduced epidermal growth factor (EGF)-induced Raf, MEK, and mitogen-activated protein kinase (MAPK) activity. This correlates with a specific inability to achieve high levels of p66(SHC) tyrosyl phosphorylation and association of p66(SHC) with GRB2, despite high levels of EGF receptor (EGFR) autophosphorylation. Notably, inhibition of EGFR or MEK/MAPK activation replaces the ECM requirement for RARbeta induction. Our results strongly suggest that a key mechanism by which specific ECMs facilitate retinoid-induced mucosecretory differentiation of NHBEs is by restricting the level of EGFR-dependent MEK/MAPK activation evoked by autocrine and/or paracrine EGFR ligands.
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PMID:Integration of growth factor, extracellular matrix, and retinoid signals during bronchial epithelial cell differentiation. 977 81

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.
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PMID:Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathways. 983 64

Cell motility is induced by many growth factors acting through cognate receptors with intrinsic tyrosine kinase activity (RPTK). However, most of the links between receptor activation and the biophysical processes of cell motility remain undeciphered. We have focused on the mechanisms by which the EGF receptor (EGFR) actuates fibroblast cell motility in an attempt to define this integrated process in one system. Our working model is that divergent, but interconnected pathways lead to the biophysical processes necessary for cell motility: cytoskeleton reorganization, membrane extension, formation of new adhesions to substratum, cell contraction, and release of adhesions at the rear. We postulate that for any given growth factor some of the pathways/processes will be actively signaled and rate-limiting, while others will be permissive due to background low-level activation. Certain couplings have been defined, such as PLCgamma and actin modifying proteins being involved in cytoskeletal reorganization and lamellipod extension and MEK being implicated in detachment from substratum. Others are suggested by complementary investigations in integrin-mediated motility, including rac in membrane protrusion, rho in new adhesions, myosin II motors in contraction, and calpain in detachment, but have yet to be placed in growth factor-induced motility. Our model postulates that many biochemical pathways will be shared between chemokinetic and haptokinetic motility but that select pathways will be activated only during RPTK-enhanced motility.
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PMID:Epidermal growth factor receptor-mediated motility in fibroblasts. 985 37

Protein kinase C (PKC) designates a family of kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human astrocytoma cell line, and their ability to block mitogen-activated protein (MAP) kinase pathway activated by substance P (SP) neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion protein by enzyme components of the MAP kinase pathway and the upstream activation of PKC (PKC activation-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity). In brief, we have demonstrated that the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced luciferase activity in this cell system is mediated via the MAP kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (PD 098059 or U0126). We also demonstrated that TPA-induced luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (CGP 41251, Go 6976, and GF 109203X) in a concentration dependent manner. In contrast, epidermal growth factor (EGF)-induced luciferase activity, which is independent of PKC activation (Ras-->Raf-1-->MEK1-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity) can only be blocked using a selective EGF receptor inhibitor (AG 1478). In conclusion, we have constructed a model cell system for the high throughput screening and identification of PKC inhibitors potentially active against astrocytoma cells in culture.
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PMID:A high throughput system for the evaluation of protein kinase C inhibitors based on Elk1 transcriptional activation in human astrocytoma cells. 991 10

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio- and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241. 82 ng/10(8) cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PLA2-AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.
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PMID:Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor. 998 14

We have tested the hypothesis that growth factor signaling pathways are augmented in hypertension, a disease associated with vascular smooth muscle cell growth. Thoracic aorta was dissected from deoxycorticosterone acetate-salt (DOCA-salt) and one kidney, one clip (1K, 1C) hypertensive rats and from sham normotensive rats for use in isolated tissue bath experiments. Systolic blood pressure was significantly higher in DOCA-salt and 1K, 1C than in normotensive sham rats: 192 +/- 7, 185 +/- 10, and 117 +/- 4 mmHg, respectively. Although virtually no contraction to epidermal growth factor (EGF) was observed in endothelium-denuded sham rat aorta [1 +/- 1% phenylephrine (PE) (10 micromol/l)-induced contraction], the maximal EGF-induced contraction was 45 +/- 7% in endothelium-denuded aorta from DOCA-salt hypertensive rats and 39 +/- 7% in aorta from 1K, 1C rats. Although slightly attenuated, a contraction to EGF was still observed in endothelium-intact aortic strips from 28-day DOCA-salt hypertensive rats. We also conducted concentration-response curves to EGF on days 1, 3, 5, 7, 14, and 21 of DOCA-salt therapy. A significant contraction to EGF in aorta from DOCA-salt rats was observed on day 14, when DOCA-salt rats had significantly higher blood pressure than sham rats: 188 +/- 6 and 122 +/- 3 mmHg, respectively. Transforming growth factor-alpha, an agonist of the EGF receptor, contracted DOCA-salt rat aorta (30 +/- 7% PE-induced contraction) but not sham aorta (3 +/- 3%). The EGF receptor tyrosine kinase inhibitor 4,5-dianilinophthalimide (10 micromol/l), the mitogen-activated protein kinase kinase inhibitor PD-098059 (10 micromol/l), and the L-type voltage-gated calcium channel inhibitor diltiazem (1 mol/l), but not the cyclooxygenase inhibitor indomethacin (10 micromol/l), virtually abolished EGF-induced contraction (85, 98, and 99% reduction, respectively). These data support a striking difference in EGF signaling between normotensive and hypertensive animals. Furthermore, they provide evidence that growth factors should be considered vasoconstrictors as well as growth modulators in hypertension.
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PMID:Epidermal growth factor: a potent vasoconstrictor in experimental hypertension. 1007 82

We studied the role of Ras signaling in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, caused excessive cell death in developing eyes at pupal stages. The Argos-induced cell death was suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppressed the Argos-induced cell death. Thus, Argos-induced cell death seemed to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid was suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death.
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PMID:Argos induces programmed cell death in the developing Drosophila eye by inhibition of the Ras pathway. 1020 Apr 72

The trefoil gene family of mucus cell-secreted proteins is a critical mediator of gastrointestinal mucosal restitution. Transcription of trefoil genes is induced during mucosal repair, but the regulatory mechanisms involved are unknown. Mice deficient in the intestine-specific peptide intestinal trefoil factor (ITF), in which colonic restitution is lethally impaired, showed reduced expression of the gastric trefoil genes SP and pS2, suggesting that trefoil peptides may individually regulate transcription of the entire family. In gastric cell lines, the trefoils were shown to act in a manner suggestive of immediate-early genes capable of auto- and cross-induction through cis-acting regulatory regions. Trefoil-mediated transcriptional regulation required activation of the Ras/MEK/MAP kinase signal transduction pathway. EGF receptor (EGF-R) activation was also necessary for trefoil auto- and cross-induction, and both spasmolytic polypeptide (SP) and ITF stimulation of gastric cell lines led to phosphorylation of EGF-R. Nevertheless, ITF and ITF-thioredoxin cell surface binding at 4 degrees C colocalized not with EGF-R, but with CD71, which is found in clathrin-coated pits, suggesting that integration of trefoil peptide responses may occur after internalization. As EGF-R expression is itself strongly induced after mucosal damage, the trefoil/EGF-R relationship may be pivotal in the generation and maintenance of the mucosal repair phenotype.
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PMID:The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor- and MAP kinase-dependent interregulation. 1022 80

Bone morphogenetic protein-2 (BMP-2) is a member of the TGFbeta superfamily of growth and differentiation factors. We investigated the effect of BMP-2 on epidermal growth factor (EGF)-induced mitogenic signaling in kidney glomerular mesangial cells. BMP-2 dose-dependently inhibits EGF-induced DNA synthesis. Maximum effect was obtained at a concentration of 100 ng/ml. BMP-2 had no inhibitory effect on the EGF receptor (EGFR)-associated tyrosine kinase activity indicating that inhibition of DNA synthesis is due to regulation of post-receptor signaling event(s). EGF stimulates MAPK activity in mesangial cells in a time-dependent manner. Inhibition of MAPK by the MEK inhibitor PD098059 blocks EGF-induced DNA synthesis indicating the requirement of this enzyme activity in EGF-mediated mitogenic signaling. Furthermore, we show that exposure of mesangial cells to BMP-2 blocks EGF-induced MAPK activity which leads to phosphorylattion of Elk-1 transcription factor. Using a GAL-4 DNA binding-domain-Elk-1 transactivation domain fusion protein-based reporter assay, we demonstrate that BMP-2 inhibits EGF-induced Elk-1-mediated transcription. These data provide the first evidence that BMP-2 signaling in mesangial cells initiates a negative regulatory cross-talk with MAPK-based transcription to inhibit EGF-induced DNA synthesis.
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PMID:Bone morphogenetic protein-2 inhibits MAPK-dependent Elk-1 transactivation and DNA synthesis induced by EGF in mesangial cells. 1032 14


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