Characterization of Dp71Δ(78-79), a novel dystrophin mutant that stimulates PC12 cell differentiation

Dp71 has an important role in the central nervous system. To better understand the function of Dp71 domains in neuronal differentiation, PC12 cells were stably transfected with a dystrophin mutant, Dp71Δ(78-79) , which lacks exons 78 and 79. Based on the percentage of cells bearing neurites and neurite length analyses, we found that cells stably expressing Dp71Δ(78-79) (PC12-C11) differentiate more efficiently than non-transfected cells. While wild-type cells reach their maximum differentiation 9-12 days after initiating the differentiation process, the PC12-C11 cells reach differentiation in 4-6 days. Protein expression analysis showed a down-regulation of Dp71a and an up-regulation of Dp71ab and/or Up71, β-dystroglycan and neuron-specific enolase in undifferentiated and in neural growth factor differentiated PC12-C11 cells. No change was observed in the expression of Grb2 and Up400. The subcellular localization of Dp71Δ(78-79) was in the cell periphery, and there was no change in localization during the differentiation process, which was also observed throughout the neurite extensions.     

Dystrophin Dp71 is required for neurite outgrowth in PC12 cells

To determine the role of Dp71 in neuronal cells, we generated PC12 cell lines in which Dp71 protein levels were controlled by stable transfection with either antisense or sense constructs. Cells expressing the antisense Dp71 RNA (antisense-Dp71 cells) contained reduced amounts of the two endogenous Dp71 isoforms. Antisense-Dp71 cells exhibited a marked suppression of neurite outgrowth upon the induction with NGF or dibutyryl cyclic AMP. Early responses to NGF-induced neuronal differentiation, such as the cessation of cell division and the activation of ERK1/2 proteins, were normal in the antisense-Dp71 cells. On contrary, the induction of MAP2, a late differentiation marker, was disturbed in these cells. Additionally, the deficiency of Dp71 correlated with an altered expression of the dystrophin-associated protein complex (DAPC) members alpha and beta dystrobrevins. Our results indicate that normal expression of Dp71 is essential for neurite outgrowth in PC12 cells and constitute the first direct evidence implicating Dp71 in a neuronal function.     

Dp71ab/DAPs complex composition changes during the differentiation process in PC12 cells

PC12 cells express different Dp71 isoforms originated from alternative splicing; one of them, Dp71ab lacks exons 71 and 78. To gain insight into the function of Dp71 isoforms we identified dystrophin associated proteins (DAPs) that associate in vivo with Dp71ab during nerve growth factor (NGF) induced differentiation of PC12 cells. DAPs expression was analyzed by RT-PCR, Western blot and indirect immunofluorescence, showing the presence of each mRNA and protein corresponding to alpha-, beta-, gamma-, delta-, and epsilon-sarcoglycans as well as zeta-sarcoglycan mRNA. Western blot analysis also revealed the expression of beta-dystroglycan, alpha1-syntrophin, alpha1-, and beta-dystrobrevins. We have established that Dp71ab forms a complex with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and alpha-, beta- and gamma-sarcoglycans in undifferentiated PC12 cells. In differentiated PC12 cells, the complex composition changes since Dp71ab associates only with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and delta-sarcoglycan. Interestingly, neuronal nitric oxide synthase associates with the Dp71ab/DAPs complex during NGF treatment, raising the possibility that Dp71ab may be involved in signal transduction events during neuronal differentiation.     

PTEN suppression promotes neurite development exclusively in differentiating PC12 cells via PI3‐kinase and MAP kinase signaling

As a dual‐specificity phosphatase catalyzing the dephosphorylation of phosphatidylinositols and protein substrates, PTEN is critically involved in the nervous system development. However, the regulatory role of PTEN in neurite outgrowth is still controversial, and the downstream signaling events remain elusive. Here, we show that PTEN knockdown promoted the proliferation and survival but not the neurite outgrowth of rat pheochromocytoma PC12 cells when exposed to nerve growth factor (NGF). In contrast, selective PTEN silencing in differentiating PC12 cells that express nestin significantly facilitated neurite elongation. Elevated Akt and Erk1/2 phosphorylation was involved in accelerated NGF‐induced neurite development of PC12 cells following PTEN knockdown. Discriminated roles of the lipid phosphatase and protein phosphatase activities of PTEN in neurite development, as well as the detailed molecular profiles affected by these phosphatase activities, were defined by restored expression of a lipid phosphatase‐deficient PTEN mutant following endogenous PTEN silencing in PC12 cells. Our study suggests an overall inhibitory effect of PTEN in neurite development reconciled by a probably indispensable role of this phosphatase in the initiation of PC12 cell differentiation. J. Cell. Biochem. 111: 1390–1400, 2010. © 2010 Wiley‐Liss, Inc.     

The A2A adenosine receptor rescues neuritogenesis impaired by p53 blockage via KIF2A,a kinesin family member

The A_2A adenosine receptor (A_2AR) is a G‐protein–coupled receptor. We previously reported that the C terminus of the A_2AR binds to translin‐associated protein X (TRAX) and modulates nerve growth factor (NGF)‐evoked neurite outgrowth in PC12 cells. Herein, we show that neuritogenesis of primary hippocampal neurons requires p53 because blockage of p53 suppressed neurite outgrowth. The impaired neuritogenesis caused by p53 blockage was rescued by activation of the A_2AR (designated the A_2A rescue effect) in a TRAX‐dependent manner. Importantly, suppression of a TRAX‐interacting protein (kinesin heavy chain member 2A, KIF2A) inhibited the A_2A rescue effect, whereas overexpression of KIF2A caused a rescue effect. Expression of a KIF2A fragment (KIF2A₅₁₄), which disturbed the interaction between KIF2A and TRAX, blocked the rescue effect. Transient colocalization of TRAX and KIF2A was detected in the nucleus of PC12 cells upon NGF treatment. These data suggest that functional interaction between KIF2A and TRAX is critical for the A_2A rescue effect. Moreover, p53 blockage during NGF treatment prevented the redistribution of KIF2A from the nucleus to the cytoplasmic region. Expression of a nuclear‐retained KIF2A variant (NLS‐KIF2A) did not rescue the impaired neurite outgrowth as did the wild‐type KIF2A. Therefore, redistribution of KIF2A to the cytoplasmic fraction is a prerequisite for neurite outgrowth. Collectively, we demonstrate that KIF2A functions downstream of p53 to mediate neuritogenesis of primary hippocampal neurons and PC12 cells. Stimulation of the A_2AR rescued neuritogenesis impaired by p53 blockage via an interaction between TRAX and KIF2A. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 604–621, 2010     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells.

In this study, we examined the role of specific protein kinase C (PKC) isoforms in the differentiation of PC12 cells in response to nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells express PKC-alpha, -beta, -gamma, -delta, -epsilon, -mu, and -zeta. For PKC-delta, -epsilon, and -zeta, NGF and EGF exerted differential effects on translocation. Unlike overexpression of PKC-alpha and -delta, overexpression of PKC-epsilon caused enhanced neurite outgrowth in response to NGF. In the PKC-epsilon-overexpressing cells, EGF also dramatically induced neurite outgrowth, arrested cell proliferation, and induced a sustained phosphorylation of mitogen-activated protein kinase (MAPK), in contrast to its mitogenic effects on control cells or cells overexpressing PKC-alpha and -delta. The induction of neurite outgrowth by EGF was inhibited by the MAPK kinase inhibitor PD95098. In cells overexpressing a PKC-epsilon dominant negative mutant, NGF induced reduced neurite outgrowth and a more transient phosphorylation of MAPK than in controls. Our results suggest an important role for PKC-epsilon in neurite outgrowth in PC12 cells, probably via activation of the MAPK pathway.     

Facilitatory roles of novel compounds designed from cyclopentenone prostaglandins on neurite outgrowth-promoting activities of nerve growth factor

Cyclopentenone prostaglandins (PGs) are known to arrest the cell cycle at the G(1) phase in vitro and to suppress tumor growth in vivo. However, their effects on neurons are unclear. Here, we report that some cyclopentenone PGs function as neurite outgrowth-promoting factors. They promoted neurite outgrowth from PC12 cells and from dorsal root ganglion explants but only in the presence of nerve growth factor (NGF). We refer to these PGs as neurite outgrowth-promoting PGs (NEPPs). Through study of the structure-function relationship of NEPP1-10 and related compounds, we found that the cross-conjugated dienone moiety of NEPPs was essential for promoting neurite outgrowth, and NEPP10 was concluded to be the best candidate for drug development. We also investigated the intracellular mechanism of the promotion by NEPPs and obtained evidence that immunoglobulin heavy chain binding protein/glucose-regulated protein 78 (BiP/GRP78) plays a role in the promotion, based on the following observations: Antisense nucleotides for BiP/GRP78 gene blocked the promotion of neurite outgrowth; BiP/GRP78 protein level increased in response to NEPPs; and overexpression of BiP/GRP78 protein by adenoviral gene transfer promoted the neurite outgrowth by NGF.     

A possible involvement of cytoplasmic Ca2+ in sodium dependency of neurite outgrowth of rat pheochromocytoma PC12 cells

The effects of extracellular Na⁺, K⁺ and Cl^? on neurite outgrowth of PC12 pheochromocytoma cells were studied. Nerve growth factor (NGF)-induced neurite formation was inhibited upon substitution of choline chloride for NaCl under normal culture conditions. It was found that neurite formation increased proportionately with the concentration of Na⁺ in medium up to 150 mM. When PC12 cells were exposed to NGF in suspension culture followed by transfer to new dishes, they showed neurite extention in response to NGF in an RNA- and protein synthesis-independent manner. Under these conditions, neurite outgrowth occurred normally in 60–150 mM Na⁺, whereas it decreased significantly at lower concentrations of Na⁺. Na⁺ dependency was also observed for cyclic AMP-mediated neurite formation of PC12 cells. In contrast, neurite outgrowth was independent of K⁺ in the range 5–106 mM, suggesting that membrane potential did not play a role in this process. No alterations were observed in neurite outgrowth with Cl^? replaced by NO^?₃, SO^2?₄, or 2-hydroxyethanesulfonate. Thus, extracellular Na⁺ plays a role in controlling neurite formation of these cells. An attempt was made to relate this effect to a decrease in cytoplasmic Ca²⁺ concentration monitored by a fluorescent dye sensitive to Ca²⁺.     

Artepillin C Derived from Propolis Induces Neurite Outgrowth in PC12m3 Cells via ERK and p38 MAPK Pathways

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 μM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.     

Cucurbitacin B induces neurogenesis in PC12 cells and protects memory in APP/PS1 mice

Cucurbitacin B (CuB) isolated from Cucumis melo by using a PC12 cell bioassay system exhibited significant nerve growth factor (NGF)‐mimic or NGF‐enhancing activity in PC12 and primary neuron cells. It was also demonstrated pro‐neurogenesis effects in ICR and APP/PS1 mice and improved memory deficit of APP/PS1 mice. Its possible mechanism includes significant induction of the phosphorylation of glucocorticoid receptor (GR), protein kinase C (PKC), phospholipase C (PLC) and inhibition of cofilin. ChemProteoBase profiling, binding assay and cellular thermal shift assay (CETSA) were used to determine the target protein. Results revealed that CuB could affect actin dynamics as an actin inhibitor but did not bind with GR. The protein level of cofilin in PC12 cells after treating 0.3 μM and different temperatures was significantly higher than that of control group. Other neurotrophic signalling pathways, such as TrkA/TrkB, were analysed with specific inhibitors and Western blot. The inhibitors of TrkA, PLC, PKC, Ras, Raf and ERK1/2 significantly decreased the percentage of PC12 cells with neurite outgrowth and shortened the length of neurite outgrowth induced by CuB. CuB significantly induced the phosphorylation of TrkA, ERK and CREB. The phosphorylation of these proteins was obviously decreased by their specific inhibitors. These results suggest that cofilin is a candidate target protein of CuB in PC12 cells and that the GR/PLC/PKC and TrkA/Ras/Raf/ERK signalling pathways play important roles in the neuroprotective effect of CuB.     

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.     

Electrical stimulation promotes nerve growth factor-induced neurite outgrowth and signaling

Background

Neurotrophins are important regulators for neural development and regeneration. Nerve growth factor (NGF) therapy has been tested in various models of neural injury and degeneration. However, whether NGF can reach target tissues and maintain effective concentration for a certain period of time remains uncertain. To facilitate neural regeneration, we investigate the possibility of combining NGF and electrical stimulation (ES) in promoting neurite outgrowth, an essential process during neural regeneration.

Methods

PC12 cells were seeded on collagen and indium tin oxide (ITO)-coated area on the transparent conductive devices. Cells were then subjected to the combination of ES and NGF treatment. Neurite outgrowth was compared.

Results

Our findings suggest that ES of 100 mV/mm together with NGF provides optimal effect on neurite outgrowth of PC12 cells. ES increases NGF-induced neurite length but reduces neurite branching, indicative of its primary effect on neurite elongation instead of initiation. One mechanism that ES enhances neurite outgrowth is through increasing NGF-induced phosphorylation of ERK1/2 (pERK1/2) and expression of Egr1 gene. ES has previously been demonstrated to increase the activity of protein kinase C (PKC). Our result indicates that activating PKC further increases NGF-induced pERK1/2 and thus neurite outgrowth.

Conclusion

It is likely that ES promotes NGF-induced neurite outgrowth through modulating the activity of ERK1/2.

General significance

Findings from this study suggest that combining ES and NGF provides a promising strategy for promoting neurite outgrowth.     

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.     

Rapid fibroblast growth factor-induced increases in protein phosphorylation and ornithine decarboxylase activity: regulation by heparin and comparison to nerve growth factor-induced increases.

Fibroblast growth factors (FGFs), like nerve growth factor (NGF), induce morphological differentiation of PC12 cells. This activity of FGF is regulated by glycosaminoglycans. To further understand the mechanisms of FGF and glycosaminoglycan actions in PC12 cells, we studied the regulation of protein phosphorylation and ornithine decarboxylase (ODC) activity by FGF in the presence and absence of heparin. As with NGF, aFGF and bFGF increased the incorporation of radioactive phosphate into the protein tyrosine hydroxylase (TH). The increase in TH phosphorylation was localized to the tryptic peptide, T3. Both T3 and T1 phosphorylations occur in response to NGF, but there was no evidence that aFGF or bFGF stimulated the phosphorylation of the T1 peptide. This result suggests differential regulation of second messenger systems by NGF and FGF in PC12 cells. Heparin, at a concentration that potentiated aFGF-induced neurite outgrowth 100-fold (100 micrograms/ml), did not alter the ability of aFGF to increase S6 phosphorylation or ODC activity. One milligram per milliliter of heparin, a concentration that inhibited bFGF-induced neurite outgrowth, also inhibited bFGF-induced increases in S6 phosphorylation and ODC activity. These observations suggest (i) that acidic and basic FGF activate a protein kinase, possibly protein kinase C, resulting in the phosphorylation of peptide T3 of TH; (ii) that the FGFs and NGF share some but not all second messenger systems; (iii) that heparin potentiates aFGF actions and inhibits bFGF actions in PC12 cells via distinct mechanisms; (iv) that heparin does not potentiate the neurite outgrowth promoting activity of aFGF by enhancing binding to its PC12 cell surface receptor; and (v) that heparin may coordinately regulate several activities of bFGF (induction of protein phosphorylation, ODC and neurite outgrowth) via a common mechanism, most likely by inhibiting the productive binding of bFGF to its PC12 cell surface receptor.     

SH2B1beta (SH2-Bbeta) enhances expression of a subset of nerve growth factor-regulated genes important for neuronal differentiation including genes encoding urokinase plasminogen activator receptor and matrix metalloproteinase 3/10

Previous work showed that the adapter protein SH2B adapter protein 1beta (SH2B1) (SH2-B) binds to the activated form of the nerve growth factor (NGF) receptor TrkA and is critical for both NGF-dependent neurite outgrowth and maintenance. To identify SH2B1beta-regulated genes critical for neurite outgrowth, we performed microarray analysis of control PC12 cells and PC12 cells stably overexpressing SH2B1beta (PC12-SH2B1beta) or the dominant-negative SH2B1beta(R555E) [PC12-SH2B1beta(R555E)]. NGF-induced microarray expression of Plaur and Mmp10 genes was greatly enhanced in PC12-SH2B1beta cells, whereas NGF-induced Plaur and Mmp3 expression was substantially depressed in PC12-SH2B1beta(R555E) cells. Plaur, Mmp3, and Mmp10 are among the 12 genes most highly up-regulated after 6 h of NGF. Their protein products [urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 3 (MMP3), and MMP10] lie in the same pathway of extracellular matrix degradation; uPAR has been shown previously to be critical for NGF-induced neurite outgrowth. Quantitative real-time PCR analysis revealed SH2B1beta enhancement of NGF induction of all three genes and the suppression of NGF induction of all three when endogenous SH2B1 was reduced using short hairpin RNA against SH2B1 and in PC12-SH2B1beta(R555E) cells. NGF-induced levels of uPAR and MMP3/10 and neurite outgrowth through Matrigel (MMP3-dependent) were also increased in PC12-SH2B1beta cells. These results suggest that SH2B1beta stimulates NGF-induced neuronal differentiation at least in part by enhancing expression of a specific subset of NGF-sensitive genes, including Plaur, Mmp3, and/or Mmp10, required for neurite outgrowth.     

Activation of Rho-Dependent Cell Spreading and Focal Adhesion Biogenesis by the v-Crk Adaptor Protein

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160^ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160^ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, α₃-integrin, and a higher-molecular-weight form of β₁-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.     

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras,or src tyrosine kinase

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.     

Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.