NGF-stimulated Neurite Outgrowth Research Articles

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

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.

The trophic responses of avian sensory ganglia in vitro to N-acetylated and des-acetyl forms of α-melanocyte stimulating hormone (α-MSH) are qualitatively distinct

α-Melanocyte-stimulating hormone (α-MSH) accelerates the regrowth of peripheral nerve axons in the rat following their transection (Verhaagen et al., Expl Neurol. 92, 451–454, 1986). The cellular mechanisms of this trophic response were investigated for several naturally occurring derivatives of α-MSH using Nerve Growth Factor (NGF)-stimulated quail sensory ganglion explants in vitro in which both neurite outgrowth and non-neuronal cell behaviour could be more reliably observed and quantified. Neurite outgrowth was determined with a semi-quantitative scoring assay. Glial migration into the outgrowth was quantified using a monoclonal antibody, GTE-52, which labels the nuclei of Schwann cells. Des-acetyl α-MSH caused a marginal increase in the neurite outgrowth density which was significant at concentrations of 0.04 and 0.1 μg/ml. The response to acetylated (N-acetyl, N,O-diacetyl) forms of α-MSH was characterized by fascicle formation by neurites which resulted in an apparent decrease in the neurite score and by the outgrowth of non-neuronal cells. Using monoclonal antibody GTE-52, which recognizes a glial nuclear antigen, these cells were identified as Schwann cells. N-Acetyl, but not des-acetyl α-MSH increased the number of GTE-52-labelled cells in the NGF-stimulated neurite outgrowth and stimulated their migration in the absence of neurites when NGF was omitted from the culture medium. Exposure of growing explants to two polyclonal antibodies against α-MSH resulted in an increased neurite outgrowth density. The results support the hypothesis that α-MSH peptides stimulate peripheral nerve growth by modulating the neurite sprouting response and demonstrate that the nature of the neurotrophic response to naturally occurring melanotropins depends on the existence of acyl substitution at the N-terminal amino acid residue. A possible role of endogenous melanotropin peptides in the regulation of sensory nerve growth is discussed.

Modulation of growth factor induced fiber outgrowth in rat pheochromocytoma (PC12) cells by a fibronectin receptor antibody

Rat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a polyclonal antiserum directed against a putative 140-kDa fibroblast cell surface fibronectin receptor (anti-gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti-gp 140 or its Fab fragments, NGF-stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS-PAGE analysis of immunoprecipitates of PC12 cells surface labeled with 125I identified a prominent 120-140-kDa band, suggesting that the site of anti-gp140 action in PC12 cells is also through a fibronectin receptor.

Metabolism via the pentose phosphate pathway in rat pheochromocytoma PC12 cells: effects of nerve growth factor and 6-aminonicotinamide.

Exposure of rat pheochromocytoma PC12 cells to 0.1 mM 6-aminonicotinamide (6AN) for 24 hours resulted in a 500-fold increase in 6-phosphogluconate indicating active metabolism of glucose via the oxidative enzymes of the pentose phosphate pathway. Amounts of 6-phosphogluconate that accumulated in 6AN-treated cells at 24 hours were significantly increased by treatment of the cells with nerve growth factor (NGF) (100 ng 7S/ml) suggesting that metabolism of glucose via the pentose pathway at this time was enhanced by NGF. This stimulation of metabolism via the pentose pathway is probably a late response to NGF because initial rates of 6-phosphogluconate accumulation in 6AN-treated cells were the same in the presence and absence of NGF. Moreover, amounts of 14CO2 generated from 1-[14CO2]glucose during the initial six hour incubation period were the same in control and NGF-treated cells. Specific activities of hexose phosphates labeled from 1-[14CO2]glucose were also the same in control and NGF-treated cells. The observation that 6AN inhibited metabolism via the pentose phosphate pathway but failed to inhibit NGF-stimulated neurite outgrowth suggests that NADPH required for lipid biosynthesis accompanying NGF-stimulated neurite outgrowth from PC12 cells can be derived from sources other than, or in addition to, the oxidative enzymes of the pentose phosphate pathway.

The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells.

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.