Inhibition of PLC-gamma1 activity converts nerve growth factor from an anti-mitogenic to a mitogenic signal in CHO cells.
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.
Zapf-Colby
A
A
Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California, CA 92093, USA.
Eichhorn
J
J
Webster
N J
NJ
Olefsky
J M
JM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases; D008934:Mitogens / Q000494:pharmacology*; D009414:Nerve Growth Factors / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C106534:FRS2 protein, human; D007527:Isoenzymes; D008565:Membrane Proteins; D008934:Mitogens; D009414:Nerve Growth Factors; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D019000:Phosphotyrosine; D007334:Insulin-Like Growth Factor I; D004247:DNA; D011494:Protein Kinases; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D051966:Phospholipase C gamma; D043265:Phosphatidylinositol Diacylglycerol-Lyase