Abstract
Nerve growth factor (NGF) causes pheochromocytoma cells (PC12) to undergo a number of physiological changes which mimic the differentiated neuronal cell, including neurite extension. We have examined protein kinase C (Ca2+/phospholipid-dependent enzyme) as a potential signaling mechanism in NGF-stimulated neurite outgrowth and induction of the enzyme ornithine decarboxylase. Phorbol 12-myristate 13-acetate (PMA) can activate protein kinase C and induce ornithine decarboxylase in PC12 cells with kinetics which are similar to those of NGF induction, but only to levels about 10-fold lower. The induction of ornithine decarboxylase by both NGF and PMA is inhibited by cycloheximide and actinomycin D suggesting that both agents increase enzyme activity by increasing gene transcription. The evidence presented here, however, shows that the induction produced by the two agents is through two different pathways. First, maximal induction by NGF is increased when PMA is included in the media showing that the two effects are synergistic. Second, NGF does not cause induction of ornithine decarboxylase in the mutant PC12nnr5 cell line (Green, S.H., Rydel, R.E., Connolly, J.L., and Greene, L.A. (1986) J. Cell Biol. 103, 1967-1978) while added PMA does produce an induction of the enzyme. Finally, when protein kinase C is down-regulated by incubating PC12 cells with PMA in serum-containing or serum-free medium for 24 h, the induction by PMA is completely inhibited, while the NGF induction is not affected. A recent study (Hall, F.L., Fernyhough, P., Ishii, D.N., and Vulliet, P.R. (1988) J. Biol. Chem. 263, 4460-4466) using sphingosine inhibition concluded that protein kinase C was required for NGF-stimulated neuritogenesis. In contrast, results presented here show that down-regulation of protein kinase C also has no effect on NGF-mediated neurite extension in PC12 cells grown in serum-free medium. Our data demonstrate that induction of ornithine decarboxylase and formation of neurites in PC12 cells by NGF does not require a protein kinase C-mediated pathway.
Highlights
Nerve growth factor (NGF) causes pheochromocy- Nerve growth factor (NGF)’ plays a pivotal role in develtoma cells (PC12) tuondergo anumber of physiological opment, maintenance, and regeneration of sensory and symchanges which mimic the differentiated neuronalcell, pathetinceurons
When proteinkinase C is down-regulated by incubating PC12 cells with Phorbol 12-myristate 13-acetate (PMA) in serum-containing or serumfree medium for 24 h, the induction by PMA is completely inhibited, while the NGF induction is not affected
Incontrast,resultspresented here show that down-regulation of protein kinase C has no effect on NGF-mediated neurite extension in PC12 cells grown in serum-free medium
Summary
Caused disruption of nearly 100% of the cells. After centrifugation, Induction of Ornithine Decarboxylase by Nerve Growth Fachalf (0.5 ml) of the supernatantwas dialyzed into phosphate-buffered saline and assayed for protein (Lowryet al., 1951)and the remainder used to determine the ornithine decarboxylase activity. 1and 2), it seemed possible that both NGF andPMA were acting throughthe same second messenger system We tested this hypothesis by determining what effect simultaneous additions of both agents had on the enzyme induction. When both stimulants were added simultaneously at saturating concentrations,the result was an induction that was larger than thesum of the inductions produced by NGF or PMA alone (Fig. 3). The effect we observed when both NGF and PMA were added is consistent with the data of End et al (1982) who showed the same phenomena in serum-containing medium These data suggest that the two stimulants may induce ornithine decarboxylase activity through different mechanisms and that the two mechanisms can act synergistically
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