Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

Abstract

The signal transduction pathways associated with neural cell adhesion molecule (NCAM)-induced neuritogenesis are only partially characterized. We here demonstrate that NCAM-induced neurite outgrowth depends on activation of p59(fyn), focal adhesion kinase (FAK), phospholipase Cgamma (PLCgamma), protein kinase C (PKC), and the Ras-mitogen-activated protein (MAP) kinase pathway. This was done using a coculture system consisting of PC12-E2 cells grown on fibroblasts, with or without NCAM expression, allowing NCAM-NCAM interactions resulting in neurite outgrowth. PC12-E2 cells were transiently transfected with expression plasmids encoding constitutively active forms of Ras, Raf, MAP kinase kinases MEK1 and 2, dominant negative forms of Ras and Raf, and the FAK-related nonkinase. Alternatively, PC12-E2 cells were submitted to treatment with antibodies to the fibroblast growth factor (FGF) receptor, inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor. Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate phosphorylation of the MAP kinases extracellular signal-regulated kinases ERK1 and ERK2. The MAP kinase activation was sustained, because ERK1 and ERK2 were phosphorylated in PC12-E2 cells and primary hippocampal neurons even after 24 hr of cultivation on NCAM-expressing fibroblasts. Based on these results, we propose a model of NCAM signaling involving two pathways: NCAM-Ras-MAP kinase and NCAM-FGF receptor-PLCgamma-PKC, and we propose that PKC serves as the link between the two pathways activating Raf and thereby creating the sustained activity of the MAP kinases necessary for neuronal differentiation.

Fig. 1.

Neuritogenic effect of NCAM on PC12-E2 cells. PC12-E2 cells were transiently transfected with pEGFP and grown for 24 hr on monolayers of fibroblasts without (a) or with (b) expression of human NCAM-140. Scale bar, 10 μm.

Fig. 2.

Effect of various compounds and signal transduction molecules on NCAM-induced neurite outgrowth. PC12-E2 cells were transiently transfected with pEGFP-N1 and an expression vector that was either empty (control) or encoding a signal transduction molecule. Alternatively, PC12-E2 cells were transfected with pEGFP-N1 alone and grown in the presence of a variety of compounds affecting signal transduction. The cells were grown for 24 hr on monolayers of fibroblasts with or without expression of human NCAM-140. Data are in all cases shown as means ± SE calculated from four or five independent experiments performed on different days. Between 200 and 300 cells were analyzed in each group in each individual experiment. The neurite length of vector-transfected cells on control fibroblasts was set to 100%, corresponding to an average value of 31 μm/cell. A, Inhibition of NCAM-stimulated neurite outgrowth from PC12-E2 cells by antibodies to FGF receptor (aFGFR) (1:1000), PLC inhibitor U-73122 (1 μm), PKC inhibitor Calphostin C (400 nm), p59^fyn inhibitor PP2 (24 μm), MEK inhibitor PD98059 (25 μm), and by expression of FRNK, dominant negative Ras (dnRas), or dominant negative Raf (dnRaf). The inhibition of NCAM-stimulated neurite outgrowth was in all cases statistically significant (p < 0.005). B, Effect of arachidonic acid (AA) (10 μm), PMA (10 ng/ml), and of constitutively active Ras (caRas), vRaf, or constitutively active MEK1 (caMEK1) and MEK2 (caMEK2). The stimulation of neurite outgrowth by the used activators was not significantly different from NCAM-induced stimulation, except for constitutively active Ras. +p < 0.05 when compared with control PC12-E2 grown on NCAM-expressing fibroblasts.

Fig. 3.

Effect of 10 μm arachidonic acid (A), 10 ng/ml PMA (B), and constitutively active MEK2 (C) on NCAM-specific neurite outgrowth affected by a series of inhibitors of signal transduction. PC12-E2 cells were transiently cotransfected with pEGFP-N1 and either an empty expression vector or one of the following expression plasmids: FRNK, dominant negative Ras, dominant negative Raf, or PC12-E2 cells transfected with pEGFP-N1 alone were grown in the presence of antibodies to the FGF receptor (diluted 1:1000), the PLCγ inhibitor U-73122 (1 μm), p59^fyn inhibitor PP2 (24 μm), the MEK inhibitor PD98059 (25 μm), or the PKC inhibitor Calphostin C (400 nm).

Fig. 4.

Stimulation of NCAM in PC12-E2 cells by the C3 peptide induces phosphorylation of ERK1 and ERK2. A, PC12-E2 cells, serum-starved (0.5% FCS) for 16 hr, were incubated in PBS without or with the C3 peptide (0.54 μm) for 7 or 40 min. Cell extracts prepared in SDS-containing lysis buffer were subjected in duplicate to SDS-PAGE and immunoblotted using either polyclonal anti-phosphoMAP kinase antibodies or polyclonal anti-MAP kinase antibodies. B, Quantification of MAP kinase phosphorylation of experiments performed as shown in A. The activity is expressed relative to the control (0 min), which was set to 100%. Error bars indicate SEs based on five independent experiments. *p < 0.05; ** p< 0.01; paired t test.

Fig. 5.

NCAM-stimulated neurite outgrowth from PC12-E2 cells or neurons is the result of sustained MAP kinase phosphorylation. Fibroblast monolayers without (LVN) or with (LBN) expression of NCAM-140 (A), cocultures of fibroblasts with or without NCAM expression and PC12-E2 cells (B,D), or cocultures of fibroblasts with or without NCAM expression and primary hippocampal neurons (C,E) were grown for 24 hr. Cell lysates were submitted to SDS-PAGE in duplicate and immunoblotted using either polyclonal anti-phosphoMAP kinase antibodies or polyclonal anti-MAP kinase antibodies. Quantification of MAP kinase phosphorylation is shown inD and E. ERK1 and ERK2 activation is expressed relative to the control (PC12-E2 or neurons cultured on monolayers of NCAM-negative fibroblasts) correcting for the total amount of ERK1 and ERK2 protein. Error bars indicate SEs based on four independent experiments. *p < 0.05; pairedt test.

Fig. 6.

A model of signaling pathways involved in NCAM-induced neurite outgrowth. Inhibitors are shown inred, and activators are shown ingreen.