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)

Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor and erythropoietin in low oxygen conditions. However, the molecular mechanisms that underlie the activation of the limiting subunit, HIF-1alpha, are still poorly resolved. Results showing that endogenous HIF-1alpha migrated 12 kDa higher than in vitro translated protein led us to evaluate the possible role of phosphorylation on this phenomenon. We report here that HIF-1alpha is strongly phosphorylated in vivo and that phosphorylation is responsible for the marked differences in the migration pattern of HIF-1alpha. In vitro, HIF-1alpha is phosphorylated by p42 and p44 mitogen-activated protein kinases (MAPKs) and not by p38 MAPK or c-Jun N-terminal kinase. Interestingly, p42/p44 MAPK stoichiometrically phosphorylate HIF-1alpha in vitro, as judged by a complete upper shift of HIF-1alpha. More importantly, we demonstrate that activation of the p42/p44 MAPK pathway in quiescent cells induced the phosphorylation and shift of HIF-1alpha, which was abrogated in presence of the MEK inhibitor, PD 98059. Finally, we found that in a vascular endothelial growth factor promoter mutated at sites previously shown to be MAPK-sensitive (SP1/AP2-88-66 site), p42/p44 MAPK activation is sufficient to promote the transcriptional activity of HIF-1. This interaction between HIF-1alpha and p42/p44 MAPK suggests a cooperation between hypoxic and growth factor signals that ultimately leads to the increase in HIF-1-mediated gene expression.
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PMID:p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1. 1055 17

Under low oxygen tension, cells increase the transcription of specific genes that are involved in angiogenesis, erythropoiesis, and glycolysis. Hypoxia-induced gene expression primarily depends on the stabilization of the alpha-subunit of hypoxia-inducible factor-1 (HIF-1alpha), which acts as a heterodimeric trans-activator. Our results indicate that stabilization of HIF-1alpha protein by treatment of proteasome inhibitors, is not sufficient for hypoxia-induced gene activation, and an additional hypoxia-dependent modification is necessary for gene expression by HIF-1alpha. Here, we demonstrate that mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD98059 does not change either the stabilization or DNA binding ability of HIF-1alpha but it inhibits the trans-activation ability of HIF-1alpha, thereby it reduces the hypoxia-induced transcription of both an endogenous target gene and a hypoxia-responsive reporter gene. We found that hypoxia induced p42/p44 mitogen-activated protein kinases (MAPKs) that are target protein kinases of MEK-1, and that expression of dominant-negative p42 and p44 MAPK mutants reduced HIF-1-dependent transcription of the hypoxia-responsive reporter gene. Our results are the first to identify that hypoxia-induced trans-activation ability of HIF-1alpha is regulated by different mechanisms than its stabilization and DNA binding, and that these processes can be experimentally dissociated. MEK-1/p42/p44 MAPK regulates the trans-activation, but not the stabilization or DNA binding ability, of HIF-1alpha.
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PMID:Mitogen-activated protein kinase kinase inhibitor PD98059 blocks the trans-activation but not the stabilization or DNA binding ability of hypoxia-inducible factor-1alpha. 1130 6

It has been suggested that blood vessel formation is an important event coupled to bone formation. The expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been shown to be greatly stimulated in osteoblasts by hypoxic stimuli such as deprivation of oxygen and treatment with cobalt. In other cell types, hypoxia-inducible factor-1 (HIF-1) that binds hypoxia-response element (HRE) has been shown to mediate gene expression induced by hypoxic stimuli. In this study, we investigated the effects of hypoxic stimuli on HIF-1, HRE, and VEGF in osteoblastic cell lines. Exposure of these cells to hypoxia or cobalt resulted in a great increase in the protein level of HIF-1alpha and the gene expression of VEGF. Transforming growth factor-beta1, prostaglandin E2, dexamethasone, and 1,25-dihydroxyvitamin D3 that have been shown to regulate VEGF gene expression in osteoblasts had no effect on HIF-1alpha induction. Blocking the enzymatic activity of phosphatidylinositol 3-kinase, p38, MEK-1 did not have any effect on the cobalt-stimulated increase of HIF-1alpha in these cells. In contrast, N-acetylcysteine (NAC), a scavenger of reactive oxygen species, abolished the cobalt induction of HIF-1alpha and that of the VEGF and a HRE-driven reporter genes. However, the hypoxia responses were not affected by NAC. These findings suggest that hypoxia and cobalt can induce VEGF gene expression in osteoblasts by increasing the level of HIF-1alpha protein through different mechanisms.
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PMID:Stabilization of hypoxia-inducible factor-1alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells. 1188 67

At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress. A common transactivator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes. HIF-1 is a heterodimeric transactivator that is composed of alpha and beta subunits. HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the alpha subunit, whereas the HIF-1beta subunit is expressed constitutively. Our previous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1alpha. In this paper, we dissected the transactivation domain of HIF-1alpha in more detail, and tested the correlation between specific domains of HIF-1alpha and specific signaling pathways. We designed several fusion proteins that contain deletion mutants of HIF-1alpha that is linked to the DNA binding domain of the yeast protein Gal4. By using the Gal4-driven reporter system, we tested the transactivation activities of the Gal4/HIF-1alpha fusion proteins in Hep3B cells. Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1alpha. We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1alpha. Treatment of PD98059, a MEK-1 inhibitor, blocked the hypoxia-induced transactivation abilities of both the 522-649 and 650-822 amino acids of the C-terminal half of HIF-1alpha. This implies that the MEK-1/p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains.
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PMID:Two transactivation domains of hypoxia-inducible factor-1alpha regulated by the MEK-1/p42/p44 MAPK pathway. 1224 58

Mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB) mediate growth and stress signals and have been implicated in the hypoxic response. Under hypoxic conditions, the expression of plasminogen activator inhibitor-1 (PAI-1) is mainly controlled by the hypoxia-inducible factor HIF-1. However, the role of MAPKs and PKB in HIF-1-mediated PAI-1 regulation is not clear. Treatment with the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, but not with the MEK1 inhibitor PD98059, abrogated hypoxia-dependent PAI-1 induction in HepG2 cells. Consistently, overexpression of PKB or of the p38 upstream kinases MKK6 and MKK3 and of JNK, but not of ERK, enhanced PAI-1 mRNA levels. In MKK3-, MKK6- and PKB-expressing cells luciferase (Luc) activities from a hypoxia-inducible PAI-1-Luc construct or from a HIF-dependent Luc construct and, concomitantly, HIF-1alpha protein levels were enhanced. These findings indicate that p38- and PKB-dependent signalling pathways contribute to enhanced PAI-1 levels in the hypoxic response.
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PMID:Regulation of the hypoxia-dependent plasminogen activator inhibitor 1 expression by MAP kinases. 1266 21

Hypoxia inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits, serves as a key regulator of metabolic adaptation to hypoxia. The amount of HIF-1alpha protein is regulated either by attenuating von Hippel-Lindau protein (pVHL)-dependent ubiquitination and subsequent 26 S proteasomal degradation or by enhancing cap-dependent mRNA translation, presumably involving a phosphatidyinositol 3-kinase (PI3K)/Akt-regulated pathway. In addition, it became apparent that Hsp90 protects HIF-1alpha from oxygen-independent degradation. Here we present evidence that PI3K/Akt is required for heat shock proteins to stabilize HIF-1alpha. In pVHL-deficient renal cell carcinoma cells, PI3K inhibition by LY294002 and wortmannin or transfection of either a dominant negative PI3K or a kinase-dead Akt mutant substantially lowered constitutively expressed HIF-1alpha without altering HIF-1alpha mRNA. Inhibitors of mitogen-activated protein kinase kinase (MAPKK) such as PD98059 or the p38 MAPK inhibitor SB203580 showed no interference. Considering that PI3K inhibitors down-regulated heat shock protein 90 (Hsp90) as well as Hsp70 expression and moreover attenuated heat- or hypoxia-induced Hsp70 as well as hypoxia-induced Hsp90 up-regulation we conclude that PI3K inhibition promoted degradation of HIF-1alpha indirectly by reducing steady state concentrations of Hsp90 and/or Hsp70. HIF-1alpha co-immunoprecipitated with Hsp90/Hsp70 and direct binding of Hsp70 to the oxygen-dependent degradation domain (ODD) of HIF-1alpha was proven by a pull-down assay and a peptide array. PI3K-mediated degradation of HIF-1alpha was confirmed in HEK 293 cells under hypoxia, suggesting that heat shock proteins constitute an integral component for HIF-1alpha accumulation. We conclude that PI3K/Akt contributes to HIF-1alpha stabilization by provoking expression of heat shock proteins.
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PMID:PI3K/Akt is required for heat shock proteins to protect hypoxia-inducible factor 1alpha from pVHL-independent degradation. 1472 29

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular adaptive responses to hypoxia. Levels of the HIF-1alpha subunit increase under hypoxic conditions. Exposure of cells to growth factors, prostaglandin, and certain nitric oxide donors also induces HIF-1alpha expression under non-hypoxic conditions. We demonstrate that muscarinic acetylcholine signals induce HIF-1alpha expression and transcriptional activity in a receptor subtype-specific manner using HEK293 cells transiently overexpressing each of M1-M4 muscarinic acetylcholine receptors. The muscarinic signaling pathways inhibited HIF-1alpha hydroxylation and degradation and induced HIF-1alpha protein synthesis that was confirmed by pulse labeling studies. Muscarinic signal-induced HIF-1alpha protein and HIF-1-dependent gene expression were blocked by treating cells with inhibitors of phosphatidylinositol 3-kinase, MAP kinase kinase, or tyrosine kinase signaling pathways. Dominant-negative forms of Ras and/or Rac-1 significantly suppressed HIF-1 activation by muscarinic signaling. Signaling via M1- and M3- but not M2- or M4-AchRs promote accumulation and transcriptional activation of HIF-1alpha. We conclude that muscarinic acetylcholine signals activate HIF-1 by both stabilization and synthesis of HIF-1alpha and by inducing the transcriptional activity of HIF-1alpha.
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PMID:Induction of hypoxia-inducible factor 1 activity by muscarinic acetylcholine receptor signaling. 1528 Mar 70

Renal proximal tubular epithelial cells (PTEC) respond to hypoxia exposure or interleukin-1beta (IL-1beta) treatment with increased vascular endothelial growth factor (VEGF) production. With respect to O2 deprivation, the hypoxia-inducible factor 1alpha/ beta (HIF-1) is the most important transcription factor driving VEGF mRNA expression. HIF-1 is also activated by IL-1beta and may thus be involved in the stimulation of VEGF production by this cytokine. However, the molecular mechanisms of HIF-1 dependent VEGF synthesis are poorly understood. Herein, human PTEC in primary culture were challenged by hypoxic incubation and/or IL-1beta treatment in absence or presence of specific phosphatidylinositol 3-kinase (PI3K) or mitogen activated protein kinase kinase-1 (MAPKK-1) inhibitors for assay of VEGF protein, VEGF mRNA and detection of HIF-1alpha by Western Blotting, EMSA and fluorescence microscopy. In addition, the activities of PI3K and MAPKK-1 were studied following hypoxia and IL-1beta treatment of the cultures. The study shows that PI3K but not MAPKK-1 inhibition resulted in the loss of hypoxic and IL-1beta induced HIF-1alpha accumulation, whereas VEGF synthesis was reduced by either intervention. Thus, PI3K signaling is required for HIF-1alpha accumulation and VEGF synthesis, whereas MAPKK-1 signaling is required for VEGF synthesis only. Furthermore, hypoxia alone was sufficient to activate PI3K in PTEC in contrast to MAPKK-1, whose activity was lowered in hypoxia.
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PMID:VEGF production by primary human renal proximal tubular cells: requirement of HIF-1, PI3-kinase and MAPKK-1 signaling. 1566 20

Hypoxia-inducible factor (HIF) is critical in the modulation of tumour angiogenesis in response to hypoxia. In the present study, the mechanisms underlying basic fibroblast growth factor (bFGF)-induced activation of HIF-1 and the subsequent release of vascular endothelial growth factor (VEGF) in a human breast cancer cell line (T47D) under normoxic conditions were explored. The data show that HIF-1alpha expression is induced by bFGF in a dose- and time-dependent fashion, while increased HIF-1alpha protein expression and transactivity of HIF-1 are due to the phosphorylation of Akt by bFGF, as indicated by application of the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. The data also show that the MEK1 (mitogen-activated protein kinase kinase-1)/ERK (extracellular signal-regulated kinase) pathway is only involved in bFGF-induced transactivity of HIF-1, but not HIF-1alpha expression, indicating roles for both the PI-3K/Akt and the MEK1/ERK pathways in bFGF activity. In addition, the translation inhibitor cycloheximide confirmed that bFGF-induced HIF-1alpha protein expression was due to de novo protein synthesis. In contrast, p38 was not required for the expression of HIF-1alpha or HIF-1 transactivity, although significant phosphorylation of p38 was observed after bFGF treatment. Treatment of the cells with bFGF increased the amount of VEGF release, and this could be suppressed by either PD98059 or LY294002, suggesting the presence of a HIF-1alpha-dependent pathway for bFGF-induced VEGF production. In conclusion, the PI-3K/Akt and MEK1/ERK pathways, in a potentially independent and co-operative fashion, can modulate HIF-1 activation by bFGF. Further studies will pinpoint whether HIF-1 is the transcriptional factor responsible for the increased VEGF production following bFGF treatment of breast tumour cells.
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PMID:In vitro study of HIF-1 activation and VEGF release by bFGF in the T47D breast cancer cell line under normoxic conditions: involvement of PI-3K/Akt and MEK1/ERK pathways. 1571 61

Tumor hypoxia is associated with cancer invasiveness, metastasis and treatment failure. Recent data suggest that the major target for endocrine treatment in breast cancer, ERalpha, is downregulated during hypoxia, but the mechanism behind this remains unknown. MAPK signaling as well as ERalpha regulation has earlier been independently linked to hypoxia and we now demonstrate HIF-1alpha and ERK1/2-activation in vivo towards the necrotic zone in DCIS of the breast, parallel with ERalpha downregulation. Hypoxia further caused transcriptional downregulation of ERalpha via activation of ERK1/2 in cell lines and, importantly, MEK1/2 inhibitors (U0126 or PD184352) or ERK1/2 suppression by siRNA partially restored the ERalpha expression. U0126 combined with tamoxifen accordingly produced an increased efficacy of the anti-estrogens during hypoxia. Based on these findings, we suggest a promising novel therapy for ERalpha-positive breast cancer where a combination of endocrine treatment and ERK1/2 inhibitors may increase treatment response by improved targeting of dormant hypoxic tumor cells.
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PMID:ERK1/2 inhibition increases antiestrogen treatment efficacy by interfering with hypoxia-induced downregulation of ERalpha: a combination therapy potentially targeting hypoxic and dormant tumor cells. 1600 58


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