The lack of a role for protein kinase C in neurite extension and in the induction of ornithine decarboxylase by nerve growth factor in PC12 cells

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.