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)

Hepatocyte growth factor (HGF) and its receptor c-Met are expressed in inappropriately high abundance in gliomas and are further upregulated during the transition from low- to high-grade malignancy. In these cells HGF induces expression of c-Met via PKC, Ras and mitogen activated protein kinase (MAPK) pathway. Here we report that secretion and expression of HGF in U87 astrocytoma is increased by a PKC activator, PMA, an effect which is abolished by a PKC inhibitor, Go6976, specific for PKCalpha and PKCbeta1. Activating PKA by forskolin, on the other hand, had no effect. Furthermore, messenger molecule downstream of PKC, i.e. MEK mediates such effect of PKC as specific MEK inhibitors (PD98059 and U0126) abolished PMA induced HGF secretion by U87 cells. Accordingly, PMA induced rapid phosphorylation of MEK substrate, i.e. Erk1/2 (p42/44 MAPK). In addition, such effect of PKC is Ras-dependent as specific Ras inhibitor L-744,832 attenuated both PMA mediated induction of Erk 1/2 phosphorylation as well as HGF secretion. Moreover, a specific p38 MAPK inhibitor (SB203580) almost completely inhibited basal HGF secretion to an undetectable level. Increased secretion of HGF is most likely exerted at the transcriptional level since inhibitor of transcription, actinomycin D abolished such increase. Furthermore, when assessed by Northern blot analysis, PMA increased HGF transcripts while U0127 and SB203580 inhibited. Therefore, our data reveal that HGF secretion in U87 cells is regulated by Ras-dependent PKC, MEK cascade and in parallel by p38 MAPK pathway. Since the Raf-PKC-MEK cascade is used for HGF's signaling via its receptor in astrocytoma cells, our data revealing similar regulatory mechanism for HGF secretion in these cells would help to explain the feed forward nature of HGF action in glioma cells that would further accentuate its basal secretion, exacerbating its effects on the progression of gliomas in an autocrine fashion.
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PMID:PKC, p42/44 MAPK and p38 MAPK regulate hepatocyte growth factor secretion from human astrocytoma cells. 1219 96

Malignant astrocytomas are characterized by extensive vascularization attributed to increased expression of the angiogenic cytokine vascular endothelial growth factor (VEGF). VEGF is elevated in astrocytomas under normal oxygen conditions and undergoes induction in hypoxic stress. Prior studies have shown that both the phosphatidylinositol 3'-kinase (PI3-kinase) and MEK1/2 (MAPK/ERK kinase 1/2) pathways promote proliferation of astrocytoma cells and growth of astrocytic tumors. Whether these pathways regulate growth by modulating angiogenesis as well as proliferation is not clear. In this study, pharmacologic inhibitors were used to specifically inhibit PI3-kinase and MEK1/2 activity in human malignant astrocytoma cell lines, and their effects on VEGF expression were determined. Northern blot analysis of VEGF messenger RNA (mRNA) from cells treated with inhibitors demonstrated cell line-specific responses. The PI3-kinase pathway regulated both the normoxic expression and hypoxic induction of VEGF in 2 cell lines, whereas MEK1/2 regulated only the normoxic expression in the same 2 lines. The third cell line showed no change in VEGF mRNA with inhibition of either of these 2 pathways. This study suggests that modulation of signaling pathways implicated in proliferation of astrocytoma cell lines may have varying effects in vivo depending on the role these pathways play in regulating tumor angiogenesis.
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PMID:Phosphatidylinositol 3'-kinase and MAPK/ERK kinase 1/2 differentially regulate expression of vascular endothelial growth factor in human malignant astrocytoma cells. 1235 54

P2Y2 receptor up-regulation and activation induces intimal hyperplasia and monocyte/macrophage infiltration in the collared rabbit carotid artery model of vascular injury, suggesting a potential role for P2Y2 receptors in monocyte recruitment by vascular endothelium. In this study, we addressed the hypothesis that activation of P2Y2 receptors by extracellular nucleotides modulates the expression of adhesion molecules on vascular endothelial cells that are important for monocyte recruitment. Results indicated that the equipotent P2Y2 receptor agonists UTP or ATP (1-100 microm) stimulated the expression of vascular cell adhesion molecule-1 (VCAM-1) in human coronary artery endothelial cells (HCAEC) in a time- and dose-dependent manner. P2Y2 antisense oligonucleotides inhibited VCAM-1 expression induced by UTP but not by tumor necrosis factor-alpha. Furthermore, UTP induced VCAM-1 expression in human 1321N1 astrocytoma cell transfectants expressing the recombinant P2Y2 receptor, whereas vector-transfected control cells did not respond to UTP. The effect of UTP on VCAM-1 expression in HCAEC was prevented by depletion of intracellular calcium stores with thapsigargin or by inhibition of p38 mitogen-activated protein kinase or Rho kinase, but was not affected by inhibitors of the mitogen-activated protein/extracellular signal-regulated kinase pathway (i.e. MEK1/2). Consistent with a role for VCAM-1 in the recruitment of monocytes, UTP or ATP increased the adherence of monocytic U937 cells to HCAEC, an effect that was inhibited by anti-VCAM-1 antibodies. These findings suggest a novel role for the P2Y2 receptor in the p38- and Rho kinase-dependent expression of VCAM-1 that mediates the recruitment of monocytes by vascular endothelium associated with the development of atherosclerosis.
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PMID:The P2Y2 nucleotide receptor mediates UTP-induced vascular cell adhesion molecule-1 expression in coronary artery endothelial cells. 1271 97

By using pharmacological and molecular approaches, we previously showed that the G-protein-coupled, extracellular calcium (Ca2+(o))-sensing receptor (CaR) regulates a large-conductance (approximately 140 pS), Ca(2+)-activated K+ channel [IK(Ca); CAKC] in U87 astrocytoma cells. Here we show that elevated Ca2+(o) stimulates extracellular-signal-regulated kinase (ERK1/2) and p38 MAP kinase (MAPK). The effect of high Ca2+(o) on p38 MAPK but not ERK1/2 is CaR mediated, insofar as transduction with a dominant-negative CaR (R185Q) using recombinant adeno-associated virus (rAAV) attenuated the activation of p38 MAPK but not of ERK1/2. p38 MAPK activation by the CaR is likely to be protein kinase C (PKC) independent, in that the pan-PKC inhibitor GF109203X failed to abolish the high-Ca2+(o)-induced phosphorylation of p38 MAPK. Consistently with our data on the activation of this kinase, we observed that inhibiting p38 MAPK blocked the activation of the CAKC induced by the specific pharmacological CaR activator NPS R-467. In contrast, inhibiting MEK1 only transiently inhibited the activation of this K+ channel by NPS R-467, despite the continued presence of the antagonist. Similarly to the lack of any effect of the PKC inhibitor on the activation of ERK1/2 and p38 MAPK, inhibiting PKC had no effect on NPS R-467-induced activation of this channel. Therefore, our data show that the CaR, acting via p38 MAPK, regulates a large-conductance CAKC in U87 cells, a process that is PKC independent. Large-conductance CAKCs play an important role in the regulation of cellular volume, so our results have important implications for glioma cell volume regulation.
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PMID:Regulation of a Ca2+-activated K+ channel by calcium-sensing receptor involves p38 MAP kinase. 1474 32

Bradykinin (BK), an inflammatory mediator, has been shown to increase the expression of proteins such as matrix metalloproteinases (MMPs) on brain cells and contributes to the pathophysiology of inflammatory responses. However, the mechanisms regulating MMP-9 expression by BK in rat brain astrocytes-1 (RBA-1) remain unclear. Here we report that the mitogen-activated protein kinase (MAPK) and NF-kappaB pathways participate in the induction of MMP-9 expression induced by BK in RBA cells. Zymographic, Western blotting, and RT-PCR analyses showed that BK increased expression of MMP-9 mRNA and protein in a time- and concentration-dependent manner. BK-induced MMP-9 mRNA and protein expression was inhibited by MEK1/2 inhibitor PD98059, PI3-K inhibitor LY294002, and NF-kappaB inhibitor helenalin. In accordance with these findings, BK-induced phosphorylation of p42/p44 MAPK and Akt and activation of NF-kappaB was attenuated by prior treatment with PD98059, LY294002, and helenalin, respectively. The effects of BK on MMP-9 expression and p42/p44 MAPK and Akt phosphorylation were inhibited by B(2) receptor antagonist Hoe 140, indicating the involvement of B(2) receptors revealed by [(3)H]-BK binding assay. Furthermore, BK-stimulated translocation of NF-kappaB into the nucleus was revealed by Western blotting and immnofluorescence staining and blocked by Hoe140, PD98059, LY294002, and helenalin. Taken together, these results suggest that in RBA cells, activation of p42/p44 MAPK and Akt cascades mediated through NF-kappaB pathway are essential for BK-induced MMP-9 gene expression. This study may provide insights into the regulation of MMP-9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation and brain astrocytoma.
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PMID:Intracellular signalings underlying bradykinin-induced matrix metalloproteinase-9 expression in rat brain astrocyte-1. 1524 11

Polybrominated diphenyl ethers (PBDEs) are an important class of flame retardants. Because of their presence in maternal milk and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible developmental neurotoxicity. Aim of the present study was to investigate the in vitro effects of PBDE-99 (2,2', 4,4', 5-pentabromodiphenyl ether) on astroglial cells (human 132-1N1 astrocytoma cells) and comparing it with those of the PCB mixture Aroclor 1254. Both PBDE-99 and Aroclor 1254 caused a concentration-dependent inhibition of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction, however, only the latter increased lactate dehydrogenase (LDH) release or cell death, assessed by the trypan blue assay. PBDE-99 caused translocation of the three protein kinase C (PKC) isozymes (alpha, epsilon, zeta) present in 132-1N1 astrocytoma cells, while Aroclor 1254 affected only PKCalpha and epsilon translocation. However, pre-incubation with the PKC inhibitor GF109203X or PKC down-regulation by the phorbol ester PMA, had minimal or no effect on PBDE-99 or Aroclor 1254-induced cytotoxicity. Similarly, the calcium chelator BAPTA-AM, the tyrosine kinase inhibitor genistein, and the MEK (mitogen activated protein kinase kinase) inhibitor PD98059 had no effect on PBDE-99 and Aroclor 1254 cytoxicity. On the other hand, the phosphatidylinositol 3 kinase (PI-3K) inhibitor LY290042 enhanced PBDE-99 toxicity, but did not affect Aroclor 1254. Because of the involvement of PI-3K in apoptotic cell death, the ability of PBDE-99 and Aroclor 1254 to induce apoptosis in astrocytoma cells was investigated. PBDE-99, but not Aroclor 1254, caused apoptotic cell death in astrocytoma cells, assessed by the TUNEL method and by Hoechst 33258 staining, via a p53 dependent mechanism. These results suggest that PBDE-99 and Aroclor 1254 exert differential cytotoxic effects on human astroglial cells.
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PMID:Differential in vitro neurotoxicity of the flame retardant PBDE-99 and of the PCB Aroclor 1254 in human astrocytoma cells. 1547 74

The transcription factor nuclear factor-kappa B (NF-kappaB) subunit p65 is phosphorylated by IkappaB kinase (IKK) at S536 in transactivation domain (TAD) 1. In this study, we investigate the presence of IKK sites in TAD2 of p65. Recombinant IKKbeta, but not IKKalpha, phosphorylated a GST-p65 substrate in which TAD1 was deleted. Mutational analysis revealed S468 as the only IKK site in TAD2. S468 phosphorylation occurred rapidly after TNF-alpha and IL-1beta in T cell, B cell, cervix carcinoma, hepatoma, breast cancer, and astrocytoma lines and in primary hepatic stellate cells as well as peripheral blood mononuclear cells. S468-phosphorylated p65 coimmunoprecipitated with IkappaBalpha, indicating that p65 is phosphorylated while bound to IkappaBalpha. Dominant negative IKKbeta or pharmacological IKK inhibition blocked S468 phosphorylation after TNF-alpha or IL-1beta, whereas dominant negative IKKalpha or inhibitors of MEK, p38, JNK, PI-3 kinase, or GSK-3 had no effect. p65S468A-reconstituted p65-/- mouse embryonic fibroblasts (MEFs) showed a small, but significant, elevation of NF-kappaB-driven luciferase activity and RANTES mRNA levels after TNF-alpha and IL-1beta in comparison to wtp65-reconstituted MEFs. p65 nuclear translocation was not altered in p65S468A-expressing MEFs. In conclusion, our results indicate that 1) IKKbeta phosphorylates multiple p65 sites, 2) IKKbeta phosphorylates p65 in an IkappaB-p65 complex, and 3) S468 phosphorylation slightly reduces TNF-alpha- and IL-1beta-induced NF-kappaB activation.
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PMID:IKKbeta phosphorylates p65 at S468 in transactivaton domain 2. 1604 71

Interleukin-1alpha (IL-1alpha) stimulates a disintegrin and metalloproteinase, ADAM-17 synthesis, consistent with activation of the soluble fragment of Amyloid Precursor Protein, APP, (sAPPalpha) in human primary astrocytes. To characterize the mechanism by which IL-1alpha promotes the non-amyloidogenic pathway of APP metabolism, we used U373 MG astrocytoma cells. IL-1alpha significantly increased levels of ADAM-10 and ADAM-17 mRNA in 16 hr. Upregulation of ADAM-17 mRNA by IL-1alpha was more pronounced despite higher basal levels of ADAM-10 mRNA. This pattern was also observed at the protein level with the upregulation of alpha-secretase. RNA interference (RNAi) of ADAM-10 and ADAM-17 inhibited IL-1alpha-stimulated sAPPalpha release and the effect was more pronounced with ADAM-17 RNAi. Concomitantly, the level of sAPPalpha was significantly increased by IL-1alpha in 48 hr; however, IL-1alpha stimulated cell-associated APP levels maximally at 6 h but the induction declined at 48 hr. IL-1alpha treatment of cells for 48 h reduced both intracellular and secreted levels of amyloid-beta, Abeta-40, and Abeta-42 peptides. Multiple MAP kinases (MAPK), including MEK/ERK, p38 kinase, PI3 kinase (PI3K) but not JNK were involved in the regulation of IL-1alpha-stimulated alpha-secretase activity and sAPPalpha release. p38 MAPK seems to be the most proximal of these MAPKs, as it was the earliest to be activated by IL-1alpha and blocking this pathway attenuated activation of IL-1alpha-induced MEK and PI3K pathways. Our data show a complex mechanism of sAPPalpha regulation by IL-1alpha that involves ADAM-10, ADAM-17 and p38 MAPK upstream of MEK and PI3K.
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PMID:Interleukin-1alpha stimulates non-amyloidogenic pathway by alpha-secretase (ADAM-10 and ADAM-17) cleavage of APP in human astrocytic cells involving p38 MAP kinase. 1672 41

Autosomal dominant Parkinson disease (PD) is caused by duplication or triplication of the alpha-synuclein gene as well as by the A30P, E46K, and A53T mutations. The mechanisms are unknown. Reactive astrocytes in the substantia nigra of PD and MPTP-treated monkeys display high levels of the inflammatory mediator intercellular adhesion molecule-1 (ICAM-1), indicating that chronic inflammation contributes to the degeneration. Here we report that alpha-synuclein strongly stimulates human astrocytes as well as human U-373 MG astrocytoma cells to up-regulate both interleukin (IL)-6 and ICAM-1 (ED50=5 microg ml(-1)). The mutated forms are more potent stimulators than wild-type (WT) alpha-synuclein in these assays. We demonstrate by immunoblotting analysis that this up-regulation is associated with activation of the major mitogen-activated protein kinase (MAPK) pathways. It is also attenuated by PD 98059, an inhibitor of the MAPK/extracellular-regulated kinase kinase MEK1/2, SP 600125, an inhibitor of c-Jun N-terminal kinase (JNK), and SB 202190, an inhibitor of p38 MAPK. The inhibitory effects on human astrocytes have IC50 values of 2, 5, and 1.5 microM respectively. We hypothesize that the neuroinflammation stimulated by release of an excess of normal alpha-synuclein or by release of its mutated forms can be involved in the pathobiology of PD.
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PMID:Alpha-synuclein and its disease-causing mutants induce ICAM-1 and IL-6 in human astrocytes and astrocytoma cells. 1701 52

Endothelin-1 (ET-1) is a potent mitogen for many cells, especially when its levels are elevated under pathological conditions, as seen in tumor cell progression and astroglial activation in neuropathies. While ET-1 is known to cause astroglial proliferation, in the present study, multiple signaling pathways involved in ET-1-mediated astrocyte proliferation were characterized. Treatment with PD98059 and U0126 (MEK inhibitors) inhibited not only ET-1-induced cell proliferation but also ET-1-activated phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in U373MG astrocytoma cells. Whereas the nonselective protein kinase C (PKC) inhibitor chelerythrine attenuated ET-1-induced cell proliferation, it was unable to block ET-1-induced ERK phosphorylation. However, ET-1 did not activate conventional or novel PKCs and did not elevate intracellular calcium. In addition, U73122 (a selective phospholipase C inhibitor), FTI-277 (an H-Ras inhibitor), as well as protein tyrosine kinase inhibitors also did not abolish ET-1-induced ERK1/2 phosphorylation. ET-1 treatment increased the activity of total Ras but not H-Ras. The phosphoinositide 3-kinase (PI3K) pathway appeared to be involved in signal transduction induced by ET-1, but it did not appear to participate in cross talk with the mitogen-activated protein kinase (MAPK) pathway. Activated ET receptors did not propagate signals either through protein tyrosine kinases or transactivation of EGF receptor tyrosine kinases, which typically trigger Ras-Raf-MAPK pathways. The results indicate that ET-1 stimulates cell proliferation by the activation of MAPK-, PKC-, and PI3K-dependent pathways that appear to function in a parallel manner. There is no apparent, direct "cross talk" between these pathways in U373MG cells, but rather, they might act on the independent but necessary components of the mitogenic effects of ET-1.
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PMID:Parallel signaling pathways in endothelin-1-induced proliferation of U373MG astrocytoma cells. 1732 70


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