Nerve growth factor and fibroblast growth factor regulate neurite outgrowth and gene expression in PC12 cells via both protein kinase C- and cAMP-independent mechanisms.

Abstract

Abstract. Nerve growth factor (NGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) promote the survival and differentiation of a variety of peripheral and central neurons. The signal transduction mechanisms that mediate the actions of these factors in neuronal cells are not well understood. We examined the effect of a deficiency in protein kinase C (PKC) and/or cAMP second messenger systems on the actions of NGF, aFGF, and bFGF in the pheoehromocytoma (PC12) cell line. Activation of PKC was not required for NGF, aFGF, and bFGF to maximally induce ornithine decarboxylase (ODC), transcription of the early response genes, d2 and d5, or neurite outgrowth. In a PC12 cell mutant that is deficient in cAMP responsiveness (A126-1B2), all three growth factors maximally induced the transcription of d5 and neurite outgrowth, but aFGF and bFGF did not induce significant increases in ODC. NGF and aFGF maximally induced the transcription of d2 in A126-1B2 cells, but bFGF-induced d2 transcription was attenuated. NGF, aFGF, and bFGF maximally induced neurite outgrowth and d5 transcription in A126 cells that were made deficient in PKC. The d2 transcriptional response was substantially reduced in cells deficient in both PKC and cAMP responsiveness. These observations lead us to conclude that (a) cAMPand PKC-dependent events are, at least in part, causally linked to NGF, aFGF, and bFGF induction of both ODC and transcription of d2 and may control functionally redundant pathways; (b) NGF, aFGF, and bFGF can elicit neurite outgrowth and increase transcription of d2 and d5 in PC12 cells via mechanisms that are independent of both PKC and cAMP; (c) NGF, aFGF, and bFGF can induce ODC in the absence of PKC; and (d) aFGF and bFGF require cAMP responsiveness to induce ODC in PC12 cells.

Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation.

“…The NGF-mediated increase in eIF-4E phosphorylation occurs in a primarily PKC-independent manner. This conclusion is consistent with the finding that NGF-promoted neuritogenesis and PC12 differentiation also occur in a PKC-independent manner (10,52) and serves to strengthen the correlation between PC12 differentiation and eIF-4E phosphorylation. Interestingly, these results differ from those reported by Morley and Traugh (46), who have demonstrated PKC dependence of eIF-4E phosphorylation in 3T3-L1 cells mitogenically stimulated with insulin.…”

“…Exposure of PC12 cells to NGF results in early transient activation of PKC (29), and persistent activation of PKC by phorbol ester induces a pattern of protein phosphorylation that is similar to that resulting from NGF treatment (9). However, down-regulation of PKC via long-term exposure of PC12 cells to high doses of PMA does not interfere with subsequent NGF-mediated differentiation or neurite outgrowth (10,52). We show here that PMA treatment of PC12 cells results in an increase in eIF-4E phosphorylation, yet to a significantly lesser extent than the increase observed upon exposure of the cells to NGF.…”

Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells.

“…Our findings that microinjection of this antibody into PC12 cells inhibits NGF-induced neurite production provides independent evidence for a role for PKC in this process. This is consistent with the observations that TPA potentiates the action of NGF in PC12 cells (Burstein et al, 1982) but appears inconsistent with the reports that NGF (and bFGF) can elicit neurite outgrowth in PC12 cells in which PKC has been down-regulated by prolonged treatment with TPA (Reinhold and Neet, 1989;Damon et al, 1990;Sigmund et al, 1990). However, down-regulating PKC in this manner may not be a totally effective means of eliminating all PKC isozymes (Cooper et al, 1989 (Curran and Morgan, 1985;Milbrandt, 1986).…”

“…This action, as well as the elimination of any constitutively expressed c-fos by autoregulatory mechanisms, may be necessary for priming in these cells, which occurs in the early phase of the NGF and bFGF response before the stimulation of any neurite outgrowth (Greene and Tischler, 1982;Rydel and Greene, 1987). The mechanisms by which NGF, bFGF, and other extracellular stimuli elicit increased transcription of cfos or other immediate early response genes are not well understood but clearly involve both PKC-dependent and -independent pathways (Cho et al, 1989;Sigmund et al, 1990;Damon et al, 1990;Altin et al, 1991a: Graham andGilman, 1991). Although tyrosine phosphorylation may be involved in these pathways, some early response genes can be induced by cAMP and by agents that elevate intracellular Ca21 (Milbrandt, 1986;Morgan and Curran, 1986;.…”

Membrane Localization of cAMP-dependent Protein Kinase Amplifies cAMP Signaling to the Nucleus in PC12 Cells

“…A126 cells are defective in importing C-PKA to the nucleus and in transcribing cAMP-induced promoters. This inhibition is specific for cAMP signals, since other stimuli (nerve growth factor or active Ras) are able to induce complete neuronal differentiation of A126 cells (11,26).…”