scholarly journals Activation of the Mitogen-Activated Protein Kinase Pathway Is Involved in and Sufficient for Megakaryocytic Differentiation of CMK Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3462-3470 ◽  
Author(s):  
Allen S. Melemed ◽  
John W. Ryder ◽  
Terry A. Vik
Keyword(s):  
Cell Surface ◽  
Map Kinase ◽  
Cell Line ◽  
Specific Inhibitor ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway ◽  
Megakaryocytic Cell

Abstract Activation of the mitogen-activated protein (MAP) kinase pathway has been associated with both cell proliferation and differentiation. Constitutively activated forms of Mek (MAP kinase/Erk kinase) and Erk (MAP kinase) have been previously shown capable of inducing differentiation or proliferation in nonhematopoietic cells. To specifically examine the role of Erk activation in megakaryocytic growth and development, we activated the MAP kinase pathway by the transfection of constitutively activated Mek or Erk cDNA into a human megakaryoblastic cell line, CMK, by electroporation. The CMK transfectant clones that expressed constitutively activated Mek or Erk showed morphologic changes of differentiation. Transfected cells also showed expression of mature megakaryocytic cell surface markers. The MAP kinase pathway was also activated by treatment of the hematopoietic cells with a cytokine that activates Erk. The treatment of CMK cells with stem cell factor (SCF ) caused MAP kinase activation and induced differentiation by the expression of mature megakaryocytic cell surface markers. The effects of the SCF treatment were inhibited by pretreatment with a specific inhibitor of the MAP kinase pathway, PD98059. In this report, we conclude that activation of the MAP kinase pathway was both necessary and sufficient to induce differentiation in this megakaryoblastic cell line.

scholarly journals Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation.

1995 ◽  
Vol 15 (7) ◽  
pp. 3644-3653 ◽  
Author(s):  
R R Vaillancourt ◽  
L E Heasley ◽  
J Zamarripa ◽  
B Storey ◽  
M Valius ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Growth Factor ◽  
Map Kinase ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway

When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.

Dibucaine and Tetracaine Inhibit the Activation of Mitogen-activated Protein Kinase Mediated by L-type Calcium Channels in PC12 Cells 

1999 ◽  
Vol 91 (6) ◽  
pp. 1798-1798 ◽  
Author(s):  
Masumi Kansha ◽  
Taro Nagata ◽  
Kazuo Irita ◽  
Shosuke Takahashi
Keyword(s):  
Map Kinase ◽  
Pc12 Cells ◽  
Local Anesthetics ◽  
Mitogen Activated Protein ◽  
Ic50 Values ◽  
Map Kinase Pathway ◽  
Kinase Signaling Pathway ◽  
Kinase Pathway ◽  
Map Kinase Signaling Pathway ◽  
Ca2 Channels

Background An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated protein (MAP) kinase signaling pathway involved in synaptic modification. It has recently been suggested that MAP kinase plays a role in coupling the synaptic excitation to gene expression in the nucleus of postsynaptic neurons. Because the effects of local anesthetics on cellular signal transduction in neuronal cells are not well-known, the authors investigated whether they affect the MAP kinase signaling pathway using PC12 cells. Methods The cells were stimulated with either 50 mM KCl or 1 microM ionomycin, and activated MAP kinase was thus immunoprecipitated. The immunocomplexes were then subjected to an Elk1 phosphorylation assay. Both the phosphorylation of MAP kinase and the induction of c-Fos were detected by immunoblotting. Results Pretreatment of the cells with 1 mM (ethylenedioxy)-diethyl-enedinitrilotetraacetic acid or 5 micron nifedipine blocked the MAP kinase activation induced by 50 mM KCl, whereas pretreatment with 2 microM omega-conotoxin GIVA did not. The expression of c-Fos induced by potassium chloride was also suppressed by dibucaine, tetracaine (concentrations that inhibited 50% of the activity of positive control [IC50s] were 16.2+/-0.2 and 73.2+/-0.7 microM, respectively), and PD 98059, a mitogen-activated/extracellular receptor-regulated kinase inhibitor. Higher concentrations of dibucaine and tetracaine were needed to suppress the activation of MAP kinase induced by ionomycin (the IC50 values of dibucaine and tetracaine were 62.5+/-2.2 and 330.5+/-32.8 microM, respectively) compared with potassium chloride (the IC50 values of dibucaine and tetracaine were 17.7+/-1.0 and 70.2+/-1.2 microM, respectively). Although probable targets of these local anesthetics might be L-type Ca2+ channels or components between Ca2+ and Ras in MAP kinase pathway, the possibility that they directly affect MAP kinase still remains. Conclusions Dibucaine and tetracaine at clinical concentrations were found to inhibit the activation of MAP kinase and the expression of c-Fos mediated by L-type Ca2+ channels in PC12 cells. The suppression of MAP kinase pathway may thus be a potential target site for the actions of dibucaine and tetracaine, including the modification of the synaptic functions.

scholarly journals The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Stimulating Expression of Toll-Like Receptor 5, CD14, MyD88, and TRIF Together with the Induction of Interleukin-8 Expression via the Mitogen-Activated Protein Kinase Pathway in Epithelial Cells

2010 ◽  
Vol 78 (5) ◽  
pp. 2153-2162 ◽  
Author(s):  
Mohamed Hafez ◽  
Kelly Hayes ◽  
Marie Goldrick ◽  
Richard K. Grencis ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Map Kinase ◽  
Interleukin 8 ◽  
Mitogen Activated Protein Kinase ◽  
Probiotic Properties ◽  
Toll Like Receptor ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway

ABSTRACT Escherichia coli strain Nissle 1917, which has been widely used as a probiotic for the treatment of inflammatory bowel disorders, expresses a K5 capsule, the expression of which is often associated with extraintestinal and urinary tract isolates of E. coli. Previously, it had been shown that the expression of a K5 capsule by Nissle 1917 was important in mediating interactions with epithelial cells and the extent of chemokine expression. In this paper, we show that infection with Nissle 1917 induces expression of Toll-like receptor 4 (TLR4) and TLR5 in Caco-2 cells and that maximal induction of TLR5 required the K5 capsule. In addition, purified K5 polysaccharide was capable of inducing expression of TLR5 and mCD14 and potentiated the activity of both TLR4 and TLR5 agonists to increase the proinflammatory response. Infection with Nissle 1917 also increased the expression of the adaptor molecules MyD88 and TRIF, which was K5 capsule dependent. By Western blot analysis, it was possible to show that induction of interleukin-8 by Nissle 1917 was predominantly through the mitogen-activated protein (MAP) kinase pathway and that expression of the K5 capsule was important for activation of the MAP kinase pathway. This paper provides new information on the function of the K5 capsule in mediating interactions between Nissle 1917 and epithelial cells and the mechanisms that underlie the probiotic properties of Nissle 1917.

Inhibition of the p38 MAP kinase pathway destabilizes smooth muscle length during physiological loading

2002 ◽  
Vol 282 (5) ◽  
pp. L1117-L1121 ◽  
Author(s):  
Oren J. Lakser ◽  
Robert P. Lindeman ◽  
Jeffrey J. Fredberg
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Muscle Length ◽  
P38 Map Kinase ◽  
Force Fluctuations ◽  
Physiological Loading ◽  
Mitogen Activated Protein ◽  
Equilibrium Length ◽  
Map Kinase Pathway ◽  
Kinase Pathway

We tested the hypothesis that mechanical plasticity of airway smooth muscle may be mediated in part by the p38 mitogen-activated protein (MAP) kinase pathway. Bovine tracheal smooth muscle (TSM) strips were mounted in a muscle bath and set to their optimal length, where the active force was maximal (Fo). Each strip was then contracted isotonically (at 0.32 Fo) with ACh (maintained at 10−4 M) and allowed to shorten for 180 min, by which time shortening was completed and the static equilibrium length was established. To simulate the action of breathing, we then superimposed on this steady distending force a sinusoidal force fluctuation with zero mean, at a frequency of 0.2 Hz, and measured incremental changes in muscle length. We found that TSM strips incubated in 10 μM SB-203580-HCl, an inhibitor of the p38 MAP kinase pathway, demonstrated a greater degree of fluctuation-driven lengthening than did control strips, and upon removal of the force fluctuations they remained at a greater length. We also found that the force fluctuations themselves activated the p38 MAP kinase pathway. These findings are consistent with the hypothesis that inhibition of the p38 MAP kinase pathway destabilizes muscle length during physiological loading.

Erk MAP kinase regulates branching morphogenesis in the developing mouse kidney

Development ◽  
2001 ◽  
Vol 128 (21) ◽  
pp. 4329-4338 ◽  
Author(s):  
Carolyn E. Fisher ◽  
Lydia Michael ◽  
Mark W. Barnett ◽  
Jamie A. Davies
Keyword(s):  
Map Kinase ◽  
Kinase Inhibitor ◽  
Branching Morphogenesis ◽  
Dependent Manner ◽  
Ureteric Bud ◽  
Strong Concentration ◽  
Erk Activation ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway

Branching morphogenesis of epithelium is a common and important feature of organogenesis; it is, for example, responsible for development of renal collecting ducts, lung airways, milk ducts of mammary glands and seminal ducts of the prostate. In each case, epithelial development is controlled by a variety of mesenchyme-derived molecules, both soluble (e.g. growth factors) and insoluble (e.g. extracellular matrix). Little is known about how these varied influences are integrated to produce a coherent morphogenetic response, but integration is likely to be achieved at least partly by cytoplasmic signal transduction networks. Work in other systems (Drosophila tracheae, MDCK models) suggests that the mitogen-activated protein (MAP) kinase pathway might be important to epithelial branching. We have investigated the role of the MAP kinase pathway in one of the best characterised mammalian examples of branching morphogenesis, the ureteric bud of the metanephric kidney. We find that Erk MAP kinase is normally active in ureteric bud, and that inhibiting Erk activation with the MAP kinase kinase inhibitor, PD98059, reversibly inhibits branching in a dose-dependent manner, while allowing tubule elongation to continue. When Erk activation is inhibited, ureteric bud tips show less cell proliferation than controls and they also produce fewer laminin-rich processes penetrating the mesenchyme and fail to show the strong concentration of apical actin filaments typical of controls; apoptosis and expression of Ret and Ros, are, however, normal. The activity of the Erk MAP kinase pathway is dependent on at least two known regulators of ureteric bud branching; the GDNF-Ret signalling system and sulphated glycosaminoglycans. MAP kinase is therefore essential for normal branching morphogenesis of the ureteric bud, and lies downstream of significant extracellular regulators of ureteric bud development.

Phagocytic Signaling Molecules in Lipid Rafts of COS-1 Cells Transfected with FcγRIIA.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2384-2384
Author(s):  
Pamela J. Mansfield ◽  
Vania Hinkovska-Galcheva ◽  
Michael Borofsky ◽  
James A. Shayman ◽  
Laurence A. Boxer
Keyword(s):  
Plasma Membrane ◽  
Map Kinase ◽  
Lipid Rafts ◽  
Specific Inhibitor ◽  
Membrane Microdomains ◽  
Signaling Proteins ◽  
Ceramide Kinase ◽  
Key Factor ◽  
Map Kinase Pathway ◽  
Kinase Pathway

Abstract FcγRIIA-mediated phagocytosis involves a number of signaling proteins and lipids, which increasingly are viewed as localizing subcellularly in plasma membrane microdomains providing a framework for their interaction. COS-1 cells stably transfected with FcγRIIA were used as a model to demonstrate co-localization of several enzymes shown to be important in polymorphonuclear leukocyte (PMN) phagocytic signaling. Previously we developed a model wherein FcγRIIA engagement in PMNs resulted in activation of phospholipase D (PLD), producing phosphatidic acid, which is hydrolyzed to diglyceride, an activator of PKC. PKCδ and Raf-1 then activate the MAP kinase pathway and subsequently myosin to allow pseudopod formation. In COS-1 cells as in PMNs, PLD in the membrane fraction was activated during phagocytosis. COS-1 PLD was found almost exclusively in lipid rafts identified by the presence of caveolin, while two of its cofactors, RhoA and ARF1, were enriched in rafts. PKCδ and Raf-1 translocated to the plasma membrane, and were enriched in lipid rafts, reaching highest levels 5 to 10 min after phagocytosis was initiated. Rottlerin, a specific inhibitor of PKCδ, completely inhibited phagocytosis, suggesting that PKCδ regulates phagocytosis in COS-1 cells; however, translocation of PKCδ to rafts was not inhibited by rottlerin. Chelation of intracellular calcium with BAPTA-AM inhibited phagocytosis by only 25%, suggesting that PKCα or PKCβ, which both require calcium, were not important regulators of phagocytosis. A specific inhibitor of MEK (which is activated by Raf and activates MAP kinase), PD098059, inhibited phagocytosis by about 35%, suggesting that the MAP kinase pathway is involved in, but not the key factor required for, COS-1 cell phagocytosis. Extracellular signal-regulated kinase-2 (ERK2), a MAP kinase, was present in the raft fraction. In PMNs, phagocytosis and activation of MAP kinase are inhibited by exogenous ceramide, and endogenous ceramide levels increase during phagocytosis, indicating that FcγRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcγRIIA that had been mutated in the ITAM region (Y282F and Y298F), rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAP kinase was activated normally, thus these processes did not require actual ingestion of particles. Previously we showed that ceramide 1-phosphate (C1P), the product of ceramide kinase (CERK), promotes membrane fusion in PMNs. Here we found that C1P increased in COS-1 cells during phagocytosis. CERK was found to be enriched in lipid rafts, translocating during phagocytosis. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.

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