Abstract
Nerve growth factor (NGF) treatment of Chinese hamster ovary fibroblast (CHO) cells exogenously expressing 2.5x105 TrkA receptors (CHO/TrkA) results in inhibition of serum and insulin-like growth factor-I (IGF-I) stimulated cell proliferation in a dose-dependent manner. Furthermore, NGF does not stimulate [3H]thymidine incorporation and inhibits IGF-I mediated DNA synthesis in CHO/TrkA cells. NGF and IGF-I induce extracellular-signal regulated kinase 1 (ERK1) and ERK2 activation, but NGF is able to stimulate a higher and more sustained activation of these enzymes compared with IGF-I. Cotreatment with NGF and IGF-I yields an ERK1/2 activity profile similar to that of NGF treatment alone. While pretreatment with mitogen activated protein kinase kinase (MKK) inhibitor PD98059 (30 microM) results in 100% inhibition of IGF-I stimulated MAPK phosphorylation (IC50<1 microM), NGF mediated MAPK phosphorylation is only decreased by 50% (IC50=3 microM). NGF, but not IGF-I, stimulates tyrosine phosphorylation and activation of PLC-gamma1 which can be inhibited in a dose-dependent manner by phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 (IC50=4 microM). Pretreatment with U73122 (IC50=7 microM) results in an 87% inhibition of NGF mediated MAPK phosphorylation, while cotreatment with PD98059 and U73122 results in 97% inhibition. U73122 pretreatment has no effect on NGF stimulated Akt activation. NGF, but not IGF-I, stimulates the tyrosine phosphorylation of Suc1-associated neurotrophic factor-induced tyrosine phosphorylation target (SNT-1)/fibroblast growth factor receptor substrate 2 (FRS2) which can be completely prevented by pretreatment with 10 microM U73122. Finally, inhibition of PI-PLC results in NGF's ability to stimulate DNA synthesis in the absence and presence of IGF-I.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing
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Animals
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CHO Cells
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cell Division / drug effects
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Cricetinae
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DNA / biosynthesis
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Enzyme Activation / drug effects
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Humans
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Insulin-Like Growth Factor I / antagonists & inhibitors
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Insulin-Like Growth Factor I / pharmacology*
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Isoenzymes / antagonists & inhibitors*
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Isoenzymes / metabolism
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Membrane Proteins / metabolism
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Mitogen-Activated Protein Kinase Kinases
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Mitogens / pharmacology*
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Nerve Growth Factors / antagonists & inhibitors
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Nerve Growth Factors / pharmacology*
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Phosphatidylinositol Diacylglycerol-Lyase
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Phosphoinositide Phospholipase C
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Phospholipase C gamma
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Phosphoproteins / metabolism
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Phosphorylation / drug effects
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Phosphotyrosine / metabolism
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Protein Kinase Inhibitors
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Protein Kinases / metabolism
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Protein Serine-Threonine Kinases*
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Receptor Protein-Tyrosine Kinases / genetics
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Receptor Protein-Tyrosine Kinases / metabolism
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Receptor, trkA
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Receptors, Nerve Growth Factor / genetics
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Receptors, Nerve Growth Factor / metabolism
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Signal Transduction / drug effects*
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Type C Phospholipases / antagonists & inhibitors*
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Type C Phospholipases / metabolism
Substances
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Adaptor Proteins, Signal Transducing
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FRS2 protein, human
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Isoenzymes
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Membrane Proteins
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Mitogens
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Nerve Growth Factors
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Phosphoproteins
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Protein Kinase Inhibitors
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Proto-Oncogene Proteins
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Receptors, Nerve Growth Factor
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Phosphotyrosine
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Insulin-Like Growth Factor I
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DNA
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Protein Kinases
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Receptor Protein-Tyrosine Kinases
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Receptor, trkA
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AKT1 protein, human
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Calcium-Calmodulin-Dependent Protein Kinases
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Mitogen-Activated Protein Kinase Kinases
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Type C Phospholipases
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Phosphoinositide Phospholipase C
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Phospholipase C gamma
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Phosphatidylinositol Diacylglycerol-Lyase