Advanced glycation endproducts stimulate the MAP-kinase pathway in tubulus cell line LLC-PK1

Abstract The DNA methyltransferase (DNMT) inhibitor decitabine increases fetal hemoglobin (HbF) in experimental primates and to therapeutic levels in patients with sickle cell disease. Decitabine-DNMT covalent adducts formed following incorporation of decitabine into newly synthesized DNA can activate DNA damage/stress response signal transduction pathways. To test the hypothesis that DNA damage/stress response pathways are involved in the ability of decitabine to increase γ-globin expression, experiments were performed in a chemical inducer of dimerization (CID)-dependent mouse fetal bone marrow (BM)-derived cell line containing the human β-globin gene locus in the context of a yeast artificial chromosome and in primary baboon erythroid progenitors derived from CD34+ BM cells grown in Iscove’s media containing 30% fetal bovine serum, 2U/ml epo, 200ng/ml SCF, and 1uM dexamethasone. Addition of decitabine (1μM) to both the CID-dependent mouse BM cell line and erythroid progenitors on day 7 of culture increased phospho-H2A.X and phospho-p38 in Western blot assays demonstrating activation of the ATM/ATR and p38 MAP kinase signal transduction pathways. Decitabine increased γ-globin 280 fold and ε-globin 38 fold in the mouse BM cell line as determined by real time PCR using the ΔΔCT method with mouse α-globin as the standard. Co-addition of the p38 MAP kinase inhibitor SB203580 reduced γ-globin induction 93.4% and ε-globin induction 88.4% in the mouse BM cell line. In primary erythroid progenitors, decitabine increased γ-globin 2.8 fold and ε-globin 44 fold. SB203580 reduced decitabine-mediated ε-globin induction 46% while no significant effect on the level of γ-globin induction was observed. Decitabine also increased phospho-CREB in the mouse BM cell line, a target of the p38 MAP kinase pathway required for γ-globin induction by histone deacetylase inhibitors. These results suggest that activation of the p38 MAP kinase pathway is required for induction of ε- and γ-globin expression by decitabine in the mouse fetal BM cell line and may contribute to increased ε-globin expression in primary erythroid progenitors. Additional Western blot analysis showed that decitabine treatment of erythroid progenitors increased p21WAF1 and p27KIP1 48–72 hours following addition of drug. Examination of Wright’s stained cytospin preparations from decitabine-treated cultured erythroid progenitors showed that orthochromatic erythroblasts were increased >2 fold and basophilic erythroblasts were decreased 5 fold compared to untreated controls 96–120 hours post-drug addition at doses ranging from 0.125 to 1.0mM. These results demonstrate that decitabine increases terminal erythroid differentiation within this cell population. As RB protein is required for cell cycle exit and terminal erythroid differentiation, we suggest that induction of terminal differentiation by decitabine is mediated by the effect of increased p21WAF1 and p27KIP1 on RB phosphorylation. Because of the known asynchrony of γ- and β-globin expression during erythroid differentiation, the ability of decitabine to induce terminal erythroid differentiation could play a role in its ability to increase γ-globin expression. We conclude that activation of DNA damage/stress response pathways by decitabine may contribute to its ability to increase γ-globin expression and induce terminal erythroid differentiation.

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Proinflammatory signal transduction pathways in N11 mouse microglial cell line activated by "Advanced Glycation Endproducts"

10.1240/sav_gbm_2002_h_000170 ◽

2002 ◽

Vol 2002 (Fall) ◽

Author(s):

Sladjana Dukic-Stefanovic ◽

Jovana Gasic-Milenkovic ◽

Winnie Deuther-Conrad ◽

Gerald M?nch

Keyword(s):

Signal Transduction ◽

Cell Line ◽

Microglial Cell ◽

Advanced Glycation Endproducts ◽

Signal Transduction Pathways ◽

Advanced Glycation ◽

Glycation Endproducts ◽

Microglial Cell Line

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

Blood ◽

10.1182/blood.v90.9.3462 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3462-3470 ◽

Cited By ~ 61

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.

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Phosphorylation of ezrin enhances microvillus length via a p38 MAP-kinase pathway in an immortalized mouse hepatic cell line

Experimental Cell Research ◽

10.1016/j.yexcr.2005.09.018 ◽

2006 ◽

Vol 312 (2) ◽

pp. 111-120 ◽

Cited By ~ 38

Author(s):

M LAN ◽

T KOJIMA ◽

M MURATA ◽

M OSANAI ◽

K TAKANO ◽

...

Keyword(s):

Map Kinase ◽

Cell Line ◽

P38 Map Kinase ◽

Hepatic Cell ◽

Hepatic Cell Line ◽

Map Kinase Pathway ◽

Kinase Pathway

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EGF inhibits muscarinic receptor-mediated calcium signaling in a human salivary cell line

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.4.c1024 ◽

2000 ◽

Vol 279 (4) ◽

pp. C1024-C1033 ◽

Cited By ~ 46

Author(s):

Bin-Xian Zhang ◽

Chih-Ko Yeh ◽

Tazuko K. Hymer ◽

Meyer D. Lifschitz ◽

Michael S. Katz

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Cell Line ◽

Muscarinic Receptor ◽

Physiological Role ◽

Muscarinic Agonist ◽

Receptor Density ◽

Map Kinase Pathway ◽

Salivary Cell ◽

Kinase Pathway

The effects of epidermal growth factor (EGF) on intracellular calcium ([Ca2+]i) responses to the muscarinic agonist carbachol were studied in a human salivary cell line (HSY). Carbachol (10−4 M)-stimulated [Ca2+]i mobilization was inhibited by 40% after 48-h treatment with 5 × 10−10 M EGF. EGF also reduced carbachol-induced [Ca2+]i in Ca2+-free medium and Ca2+ influx following repletion of extracellular Ca2+. Under Ca2+-free conditions, thapsigargin, an inhibitor of Ca2+ uptake to internal stores, induced similar [Ca2+]i signals in control and EGF-treated cells, indicating that internal Ca2+ stores were unaffected by EGF; however, in cells exposed to thapsigargin, Ca2+influx following Ca2+ repletion was reduced by EGF. Muscarinic receptor density, assessed by binding of the muscarinic receptor antagonistl-[benzilic-4,4′-3HCN]quinuclidinyl benzilate ([3H]QNB), was decreased by 20% after EGF treatment. Inhibition of the carbachol response by EGF was not altered by phorbol ester-induced downregulation of protein kinase C (PKC) but was enhanced upon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of the carbachol response by EGF were both blocked by the MAP kinase pathway inhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca2+ release from internal stores and also exerts a direct inhibitory action on Ca2+ influx. A decline in muscarinic receptor density may contribute to EGF inhibition of carbachol responsiveness. The inhibitory effect of EGF is mediated by the MAP kinase pathway and is potentiated by a distinct modulatory cascade involving activation of PKC. EGF may play a physiological role in regulating muscarinic receptor-stimulated salivary secretion.

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