Iron enhances NGF-induced neurite outgrowth in PC12 cells

Rat pheochromocytoma 12 (PC12) cells undergo neuronal differentiation in response to nerve growth factor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular signal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting effect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF-induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.     

Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor,thereby modulating activation of MAP kinase,Akt, and neurite outgrowth in PC12 cells

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.     

The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines.

The trk tyrosine kinase proto-oncogene product gp140prototrk binds nerve growth factor (NGF) and is rapidly and selectively activated by this neurotrophic factor. To determine whether gp140prototrk is involved in transducing a functional NGF signal, PC12 cell mutants (PC12nnr) deficient in high affinity NGF binding and unresponsive to NGF were used. Northern analysis revealed that these mutant cells have greatly reduced levels of trk expression. PC12nnr cultures were transiently transfected with expression vectors encoding the full-length rat trk cDNA and assessed for responsiveness to NGF. Expression of exogenous trk rescued the capacity for NGF-promoted neurite outgrowth, cellular hypertrophy, and serum-free survival by these cells. These results indicate that gp140prototrk is necessary for functional NGF signal transduction.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Mimicry and Inhibition of Nerve Growth Factor Effects: Interactions of Staurosporine, Forskolin, and K252a in PC12 Cells and Normal Rat Chromaffin Cells In Vitro

The structurally similar compounds staurosporine and K252a are potent inhibitors of protein kinases. K252a has previously been reported to inhibit most or all of the effects of nerve growth factor (NGF) on PC12 pheochromocytoma cells, and staurosporine has been reported both to inhibit and to mimic NGF-induced neurite outgrowth from a PC12 cell subclone in a dose-dependent manner. We have studied the interactions of these agents with each other, with NGF, and with forskolin, an activator of adenylate cyclase, on the parent PC12 cell line and on normal neonatal and adult rat chromaffin cells. Staurosporine alone or in conjunction with forskolin induces outgrowth of short neurites from PC12 cells but does not substitute for NGF in promoting cell survival. It does not abolish NGF-induced neurite outgrowth but does reverse the effects of NGF on catecholamine synthesis. K252a abolishes NGF-induced neurite outgrowth but only partially decreases outgrowth induced by NGF plus forskolin. It does not inhibit neurite outgrowth produced by staurosporine or staurosporine plus forskolin. These findings with PC12 cells suggest that staurosporine might act downstream from K252a and NGF on components of one or more signal transduction pathways by which NGF selectively affects the expression of certain traits. Both neonatal and adult rat chromaffin cells show dramatic flattening and extension of filopodia in response to staurosporine, an observation suggesting that some of the same pathways might remain active in cells that do not exhibit a typical NGF response. Only a small amount of neurite outgrowth is observed, however, and only in neonatal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Trifluoromethyl Analog of Verbenachalcone Promotes Neurite Outgrowth and Cell Proliferation of NeuroScreen-1 Cells

Past research has shown that natural products of plant and marine origins and their congeners enhance the actions of neuritogenic factors of the central nervous system (CNS) such as nerve growth factor (NGF). However, the role of fluorine substitutions in their structure–activity relationship (SAR) has not been explored. We have synthesized a trifluoromethyl analog of verbenachalcone (VC), a pharmacologically active natural compound previously shown to potentiate NGF activity. This analog, designated C278, enhances neurite outgrowth and proliferation of NeuroScreen-1™ (NS-1) cells, a subclone of PC12 pheochromocytoma cells. C278 increases the percentage of neurite bearing cells in the presence of suboptimal doses of NGF in comparison with controls treated with NGF alone. In addition, C278 stimulates cell growth in reduced serum and serum-free cell culture conditions based on our observation of increases in cell number and metabolic assessment with MTT reduction and resazurin assays. The addition of C278 partially restored inhibition of NGF-induced neurite outgrowth by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126. Short-term sequential exposure of cells to U0126, C278, and NGF enhanced phosphorylation of extracellular signal-regulated kinase (ERK) in comparison with cells treated with only the MEK inhibitor and NGF. C278 also attenuated cell growth arrest caused by exposure to PD98059, U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002 but did not alter phosphorylation of Akt, a classic downstream target of PI3K during cell survival. These data suggest that C278 promotes NGF-dependent neurite outgrowth in NS-1 cells through a MEK signaling pathway by a mechanism that alters short-term activation of ERK. In contrast, C278 promotes PI3K-mediated survival independently of Akt phosphorylation.     

Fate of Cyclic Nucleotides in PC12 Cell Cultures: Uptake, Metabolism, and Effects of Metabolites on Nerve Growth Factor-Induced Neurite Outgrowth

The fate of cyclic AMP (cAMP), dibutyryl-cAMP (Bt2-cAMP), and the (Sp)-isomer of adenosine 3',5'-monophosphorothioate [(Sp)-cAMPS] was studied in the PC12 culture medium by means of HPLC. In the absence of PC12 cells, cAMP and Bt2-cAMP were rapidly degraded by nonspecific esterases and cyclic nucleotide phosphodiesterase both originating from the serum commonly used as a culture medium ingredient, whereas (Sp)-cAMPS was completely stable. Since 5'-AMP, adenosine, inosine, and hypoxanthine appeared in the culture medium after incubation with cAMP or Bt2-cAMP, we have determined their effect on nerve growth factor (NGF)-induced neurite outgrowth. 5'-AMP, adenosine, and inosine were indeed potent agents in producing a potentiating effect on NGF-induced early neurite outgrowth at a concentration of 1 mM. Thus, cAMP metabolites had the capacity to induce an effect that has been described as cAMP-specific. In serum-free culture medium and in the presence of cells, all cyclic nucleotides were taken up by PC12 cells. Uptake was highly correlated with the hydrophobic nature of the compounds, and was accompanied by a simultaneous excretion of metabolites. On incubation with cAMP, NGF had a pronounced effect on the metabolic pattern found in the culture medium. In particular, dephosphorylation of 5'-AMP was specifically enhanced. This effect of NGF on the degradation of cAMP was also apparent when cAMP metabolites were incubated with PC12 cells. Whereas 5'-AMP degradation was greatly increased, NGF had no effect on the metabolism of the other purine compounds.     

Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity

Past studies have shown that serum-free cultures of PC12 cells are a useful model system for studying the neuronal cell death which occurs after neurotrophic factor deprivation. In this experimental paradigm, nerve growth factor (NGF) rescues the cells from death. It is reported here that serum-deprived PC12 cells manifest an endonuclease activity that leads to internucleosomal cleavage of their cellular DNA. This activity is detected within 3 h of serum withdrawal and several hours before any morphological sign of cell degeneration or death. NGF and serum, which promote survival of the cells, inhibit the DNA fragmentation. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases in vitro, suppresses the endonuclease activity and promotes long-term survival of PC12 cells in serum-free cultures. This effect appears to be independent of macromolecular synthesis. In addition, ATA promotes long-term survival of cultured sympathetic neurons after NGF withdrawal. ATA neither promotes nor maintains neurite outgrowth. It is hypothesized that the activation of an endogenous endonuclease could be responsible for neuronal cell death after neurotrophic factor deprivation and that growth factors could promote survival by leading to inhibition of constitutively present endonucleases.     

Vitronectin is the major serum protein essential for NGF-mediated neurite outgrowth from PC12 cells.

The rat pheochromocytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf serum (FCS). Using a novel assay procedure we have developed a purification protocol which has allowed the isolation of the protein in serum responsible for neurite outgrowth after NGF treatment. FCS has been fractionated using four chromatographic procedures and in each case the peak of biological activity copurified with vitronectin. We have concluded, therefore, that vitronectin is the protein present in FCS which can mediate NGF-dependent neurite outgrowth in PC12 cells. Vitronectin and fibronectin from FCS have been chromatographically separated and only the former is capable of inducing neurite outgrowth. We have also shown that vitronectin utilizes the RGD amino acid sequence in binding to the surface of PC12s.     

v-Crk, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Biol. 14: 1964 - 1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native, v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 h of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 70 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation, since naive undifferentiated v-Crk expressing PC12 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk expressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when incubated for 2 weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6 - 8 h after NGF deprivation. Thus, unlike v-Crk-mediated augmentation of differentiation, sustained activation of MAP kinase may not be required for v-Crk-induced cell survival.     

Evidence for Nerve Growth Factor-Potentiating Activities of the Nonpeptidic Compound SR 57746A in PC12 Cells

Abstract: SR 57746A {1-[2-(naphth-2-yl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6-tetrahydropyridine hydrochloride} exhibits neurotrophic activities in vivo and in vitro. We used the rat pheochromocytoma PC12 cell line to investigate in vitro cellular changes induced by SR 57746A. A significant increase in the percentage of cells bearing neurite-like processes was obtained in cells treated by SR 57746A and nerve growth factor (NGF) compared with NGF treatment alone. SR 57746A added alone, however, had no effect on morphogenesis or on survival of cells in serum-free medium. In contrast, SR 57746A induced a "priming" effect on PC12 cells for neurite outgrowth within 6 h of addition of the protein tyrosine kinase inhibitor genistein. An increase in α-actinin content resulted from treatment with SR 57746A. Expression of NGF-mediated acetylcholinesterase and choline acetyltransferase was enhanced within 5 days by SR 57746A. The molecule also induced rapid F-actin redistribution. Within 2 min of incubation, outgrowth of F-actin-containing filopodia was clearly visible at the cell periphery, as previously shown with NGF. It is interesting that this effect of SR 57746A could be mimicked by protein tyrosine kinase inhibitors and abolished by preincubation with sodium orthovanadate, a protein tyrosine phosphatase inhibitor.     

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.     

Effects of Schwann cell secreted factors on PC12 cell neuritogenesis and survival

We have used PC12 cells to examine the effects of factors secreted by Schwann cells that promote cell survival and neurite outgrowth, and hence are likely candidates for promoting neuronal regeneration. RT-PCR showed that primary Schwann cells produced a range of neurotrophins, excluding NT3, but this profile was different from either of two cell lines SCTM41 or PVGSCSV40T, or forskolin-expanded Schwann cells. The effects of Schwann cell conditioned media on neurite outgrowth was tested against a range of factors, and showed clear neuritogenic effects. Of the factors tested, only NGF had a significant response on neuritogenesis. Western blotting for neurofilaments showed that primary Schwann cells induced a strong response close to that of NGF. The Trk tyrosine kinase inhibitor K252a did not block the neuritogenic effects of primary Schwann cells. In contrast, K252a blocked both NGF and the SCTM41 cell effects. Schwann cell conditioned media also enhanced PC12 cell survival. Again, in contrast with NGF or SCTM41 cells, the primary Schwann cell effect was Trk tyrosine kinase independent. The Schwann cell conditioned medium contains a protein factor (greater than 12 kDa and broken down by trypsin treatment) with remarkable thermal stability (unaffected at 95 degrees C for 15 min) and the ability to bind heparin. Our results provide clear evidence that Schwann cells produce factors other than those already known to stimulate a neural phenotype in PC12 cells, and which thus have potential regeneration enhancing effects.     

Nerve growth factor-induced neurite formation in PC12 cells is independent of endogenous cellular gangliosides

The PC12 rat pheochromocytoma cell line is an established model for nerve growth factor (NGF)-induced neurite formation. It has been shown that when gangliosides are added to the culture medium of PC12 cells, NGF-induced neurite formation of PC12 cells is enhanced. To determine the role of endogenous cellular gangliosides themselves in NGF-elicited neurite formation, we depleted cellular gangliosides using the new specific glucosylceramide synthase inhibitor, d, l-threo-1-phenyl-2- hexadecanoylamino-3-pyrrolidino-1-propanol.HCl (PPPP). 0.5-2 microM PPPP rapidly inhibited ganglioside synthesis and depletedcellular gangliosides. Nonetheless, over a concentration range of 5-100 ng/ml NGF, in both low serum and serum-free medium, neurite formation was normal. Even pretreatment of PC12 cells for up to 6 days with 1 microM PPPP followed by cotreatment with PPPP and NGF for 10 days, still did not inhibit neurite formation. The conclusion that ganglioside depletion did not block neurite formation stimulated by NGF was supported by the lack of effect of PPPP, under these same conditions, on cellular acetylcholine esterase activity, a neuronal differentiation marker (73.8 +/- 12.1 versus 67.2 +/- 4.6 nmol/min/mg protein at 50 ng/ml NGF; control versus 1 microM PPPP). These findings, together with previous studies showing enhancement of NGF-induced neurite formation by exogenous gangliosides, underscore the vastly different effects that exogenous gangliosides and endogenous gangliosides may have upon cellular functions.      

Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells

The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.     

Monokine induced by interferon-gamma acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons

We found that a monokine induced by interferon-gamma (Mig, CXCL9), which belongs to the CXC chemokine subfamily, acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons. PC12 cells were shown to express a single class of high affinity binding sites for Mig (670 receptors/cell, Kd = 2.9 nm). Mig induced neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of extracellular signal-regulated kinase signaling pathways between Mig and nerve growth factor (NGF) revealed that these pathways are crucial for Mig action as well as NGF. K252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks) did not inhibit the action of Mig, suggesting that Mig action occurs via a different receptor from that of NGF. Furthermore, Mig as well as NGF promoted PC12 survival under serum-free conditions and activated Akt/protein kinase B downstream from phosphatidylinositol 3-kinase (PI3K). Because the PI3K inhibitor LY294002 prevented the Mig- and NGF-induced survival effect, this effect is probably mediated by the PI3K signaling pathway. Mig also promoted survival of rat primary sympathetic neurons that die when deprived of NGF. These results suggest that chemokines, including Mig (CXCL9) have neurotrophic effects on the nervous system.     

Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system

The rat pheochromocytoma PC12 cell line has been an invaluable model system for studying neuritogenesis. Nerve growth factor (NGF) elicits multiple aspects of neurite outgrowth in PC12 cells. It is therefore difficult to dissect and assign an individual signaling pathway to each stage of neuritogenesis. We have recently reported the isolation of a variant PC12 cell line, PC12-N1 (N1), which spontaneously extends neuritic processes and exhibits an increased sensitivity to NGF. Here, we show that, under different culture conditions, the cells display three distinct phases of neuritogenesis consisting of neurite initiation, rapid neurite elongation, and a maturation process characterized by the thickening of neurites and increase in cell soma sizes. We demonstrate that signaling through ERK, but not p38 or JNK, is required for the spontaneous neurite initiation and extension. Treatment with low concentrations of NGF induces rapid neurite elongation without affecting neurite branching and cell soma sizes. Such a rapid neurite outgrowth can be blocked by the inhibition of ERK, but not JNK, activities. In the presence of higher concentrations of NGF, the N1 cells undergo further differentiation with many characteristics of mature neurons in culture, e.g. larger cell soma and numerous branches/connections. This process can be completely blocked by inhibiting ERK or JNK activities using specific inhibitors. These results suggest that ERK and JNK signals play different roles in neuritogenesis, and that JNK activity is essential in the late stages of neuritogenesis. Furthermore, our results demonstrate that signaling dosage is important in the activation of a specific pathway, leading to distinctive biological outcomes.     

The changes in the neuronal PC12 and the intestinal epithelial Caco-2 cells during the coculture. The functional analysis using an in vitro coculture system

The interaction between intestinal epithelial cells andperipheral neuronal cells were examined using an invitro coculture system. Two cell lines, Caco-2 and PC12, were usedfor this experiment as an intestinal epithelial and entericneuronal cell model, respectively. By coculturing with fullydifferentiated Caco-2 cells, the neurite outgrowth was inducedin PC12 cells. This neurite outgrowth in PC12 was blocked byanti-nerve growth factor (NGF) polyclonal antibodies,suggesting that the neurite outgrowth in PC12 during thecoculture with Caco-2 cells was due to NGF secreted fromCaco-2 cells. On the other hand, coculturing with fullydifferentiated PC12 cells induced the decrease oftransepithelial electrical resistance in Caco-2 cellmonolayers. The permeability of lucifer yellow alsosignificantly increased, suggesting that the barrier functionand paracellular permeability of Caco-2 monolayers werealtered by coculturing with PC12 cells. The present studysuggests that this in vitro coculture system is a good modelfor the functional analysis of interaction among intestinalepithelial cells with different cell types.     

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

To define the molecular bases of growth factor-induced signal transduction pathways, antibodies known to block the activity of either protein kinase C (PKC) or the fos protein were introduced into PC12 cells by microinjection. The antibody against PKC significantly inhibited neurite outgrowth when scored 24 h after microinjection and exposure to nerve growth factor (NGF). Microinjection of antibodies to fos significantly increased the percentage of neurite-bearing cells after exposure to either NGF or basic fibroblast growth factor (bFGF) but inhibited the stimulation of DNA synthesis by serum, suggesting that in PC12 cells, fos is involved in cellular proliferation. Thus, activation of PKC is involved in the induction of neurite outgrowth by NGF, but expression of the fos protein, which is induced by both NGF and bFGF, is not necessary and inhibits neurite outgrowth.