Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line

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 NO3-, SO2-4, 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+.     

Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.     

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⁺.     

Depolarization-Induced Increase in Surface Binding and Internalization of 125I-Nerve Growth Factor by PC 12 Pheochromocytoma Cells

The binding and internalization of 125I-nerve growth factor (NGF) by PC12 pheochromocytoma cells was studied as a function of extracellular potassium concentration. Both surface-bound and internalized fractions of 125I-NGF associated with the cells under depolarizing conditions (50 mM K+) increased to 144 +/- 28% (average +/- SEM, six different cell preparations) and to 176 +/- 12% (n = 6), respectively, of those observed at 6.0 mM K+. Scatchard-type analysis of the data indicates increased sites for the binding and internalization of iodinated NGF by the cells. Similar enhancement was observed for cells treated with NGF as well. This voltage-dependent phenomenon was reversible, and also observed in the presence of veratridine. Moreover, withdrawal of extracellular Ca2+ abolished high K+-induced modulation of 125I-NGF binding and internalization, indicating that this effect may be mediated by Ca2+.     

Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.     

Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata

We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line. In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil. Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin. With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin. With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+. Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata. The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin. Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA. Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation. These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.     

Differential action of nerve growth factor, cyclic AMP and neurotropin on PC12h cells

Nerve growth factor (NGF) induced the activities of acetylcholinesterase (AChE) and Na+,K+-ATPase concomitant with neurite outgrowth in PC12h cells, while dibutyryl cyclic AMP (DBcAMP) caused the induction of AChE activity and neurite outgrowth but not Na+,K+-ATPase activity. A nonproteinaceous extract isolated from the inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin) induced neurite outgrowth and cell surface change similar to NGF without affecting AChE activity. The results suggest that NGF, DBcAMP and Neurotropin act on PC12h cells through different mechanisms.     

Nerve growth factor-induced neurite outgrowth is potentiated by stabilization of TrkA receptors

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.     

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.     

N42－A对神经生长因子诱导PC12细胞分化的抑制作用

研究表明,缺乏神经生长因子（ＮＧＦ）的营养支持是Ａｌｚｈｅｉｍｅｒ＇ｓ等神经元退行性疾病发生发展的重要原因,而ＮＧＦ和／或ＮＧＦ受体的过度表达则与一些神经系统肿瘤的发生发展有着十分密切的因果关系。采用（１２５）Ⅰ－ＮＧＦ受体特异结合实验作为ＮＧＦ受体活性物质筛选实验模型从中药牛膝中筛选出了能强烈地抑制（１２５）Ⅰ－ＮＧＦ受体结合的活性成分Ｎ４２－Ａ（ⅠＣ（５０）＝６．１８±３．４３,ｎ＝４）;细胞培养实验表明,Ｎ４２－Ａ对ＮＧＦ诱导大鼠嗜铬神经瘤ＰＣｌ２细胞的分化也具有很强的剂量依赖性抑制作用（对０．１ｎｍｏｌ／Ｌ和０．２ｎｍｏｌ／ＬＮＧＦ诱导的大鼠嗜铬神经病ＰＣ１２细胞轴突生长的半数抑制浓度分别为６μｇ／ｍＬ和２１μｇ／ｍＬ）。这表明,Ｎ４２－Ａ是神经元上介导ＮＧＦ诱导轴突生长的特异受体抑制剂,不仅对ＮＧＦ及其受体过度表达所致的神经系统肿瘤的防治具有潜在的应用价值,而且对Ａｌｚｈｅｉｍｅｒ＇ｓ等神经元退行性疾病防治药物的开发研究具有十分重要的意义。     

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.     

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.     

Suramin Induces Phosphorylation of the High-Affinity Nerve Growth Factor Receptor in PC12 Cells and Dorsal Root Ganglion Neurons

Abstract: Suramin is a polysulfonated naphthylurea with demonstrated antineoplastic activity. Toxicity includes adrenal insufficiency and peripheral neuropathy. Although the mechanism of antitumor activity is unknown, inhibition of binding of growth factors to their receptors has been suggested. Growth factors inhibited by suramin include platelet-derived growth factor, fibroblast growth factor, transforming growth factor, epidermal growth factor, insulin-like growth factor, and nerve growth factor (NGF). In these studies, suramin was shown to be cytotoxic to PC12 cells in a dose-dependent manner. At lower doses and in surviving cells, we observed the induction of neurite outgrowth. To determine the mechanism of suramin-induced neurite outgrowth, PC12 cells were exposed to suramin and/or NGF for various time periods and treated cells were analyzed, by western blot analysis, for expression of tyrosine phosphoproteins. There was a similarity in the pattern of tyrosine-phosphorylated proteins in PC12 cells stimulated with suramin or NGF. Of particular interest was the rapid phosphorylation (by 1 min) of the high-affinity NGF (TrkA) receptor. Activation of other members of the signal-transduction cascade (Shc, p21 ^ras , Raf-1, ERK-1) revealed similar phosphorylation levels induced by suramin and NGF. Parallel studies were performed in rat dorsal root ganglion cultures; suramin potentiated neurite outgrowth and activated the NGF receptor on these cells. This finding of specific patterns of tyrosine phosphorylation of cellular proteins in response to suramin treatment demonstrated that suramin is a partial agonist for the NGF receptor in both PC12 cells and dorsal root ganglion neurons.     

NGF promotes copper accumulation required for optimum neurite outgrowth and protein methylation

The role of copper in biological phenomena that involve signal transduction is poorly understood. A well-defined cellular model of neuronal differentiation has been utilized to examine the requirement for copper during nerve growth factor (NGF) signal transduction that results in neurite outgrowth. Experiments demonstrate that NGF increases cellular copper content within 3 days of treatment. Copper chelators reduce the effects of NGF on neurite outgrowth and copper accumulation. The effects of tetraethylene pentamine (TEPA), a copper-specific chelator, are reversible by removal from the culture medium and/or by addition of equimolar copper chloride. Because previous work demonstrated that NGF increases protein methylation in PC12 cells, we examined whether TEPA also inhibits S-adenosylhomocysteine hydrolase (SAHH), an essential copper enzyme involved in all protein methylation reactions. In addition to direct in vitro inhibition of SAHH, we show that TEPA decreases protein arginine methyltransferase 1(PRMT1)-specific enzyme activity in PC12 cells and sympathetic neurons. These data comprise the first biochemical and cellular evidence to address the mechanism of copper involvement in neuronal differentiation.     

Protective effect of neurotrophic factors, neuropoietic cytokines and dibutyryl cyclic AMP on hydrogen peroxide-induced cytotoxicity on PC12 cells: a possible link with the state of differentiation

We present evidence that the survival of PC12 cells exposed to hydroxyl radicals generated by hydrogen peroxide applied for 30 min at 1 mM was effective when they were differentiated in response to Nerve Growth Factor (NGF) and/or other inducers of neurite outgrowth such as basic-fibroblast growth factor and dibutyryl cyclic AMP. The time- and dose-dependent differentiation triggered by NGF was (1) markedly increased by basic fibroblast growth factor, interleukin-6 or dibutyryl cyclic AMP; (2) diminished by leukemia inhibitory factor or ciliary neurotrophic factor; (3) not potentiated by insulin-like growth factor I or progesterone. The influence of these various factors and agents on PC12 cells was evaluated by the estimation of neurite outgrowth, whereas their possible protective effects were assessed by the measurement of cell survival. Our results would indicate that the factors and agents that induced differentiation were also able to protect the cells against an oxidative stress.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

Protein Kinase Inhibitor H-7 Differentially Affects Early and Delayed Nerve Growth Factor Responses in PC12 Cells

Abstract: The effects of the protein kinase inhibitor H-7 on early and delayed responses to nerve growth factor (NGF) were investigated in PC12 cells. H-7 reduced the NGF-induced expression of c-Fos in a dose-dependent manner without affecting the time course of c-Fos appearance. Conversely, H-7 potentiated delayed NGF effects, i.e., neurite outgrowth and Ca²⁺/phospholipid-dependent protein kinase (PKC) induction, but not choline acetyltransferase induction. Long-term treatment with NGF resulted in an increase of at least four tyrosine-phosphorylated protein bands with molecular masses between 39 and 48 kDa, which was also potentiated by H-7. In the absence of NGF, H-7 had no significant effect on c-Fos expression, tyrosine phosphorylation of the 45 kDa protein, or choline acetyltransferase activity. However, 4 days of exposure to H-7 alone induced PKC activity and tyrosine phosphorylation of the 39-kDa protein. The action of H-7 derivatives on neurite outgrowth did not correlate with their inhibition profile of cyclic nucleotide-dependent protein kinases. Down-regulation of PKC activity by prolonged exposure to phorbol ester did not completely abolish the effects of NGF and H-7 on induction of c-Fos, choline acetyltransferase activity, and neurite outgrowth, indicating that PKC-independent pathways contribute to these actions. These results suggest that additional pathway(s) sensitive to H-7 may exist, which induce immediate early gene expression and suppress neuronal differentiation of PC12 cells.     

Nitric oxide underlies the differentiation of PC12 cells induced by depolarization with high KCl

Nitric oxide (NO) acts as a cytostatic agent to induce neuronal differentiation of PC12 cells after nerve growth factor (NGF) treatment. We newly subcloned PC12K cells that extended neurites after depolarization with high KCl. Here we present evidence that the neuronal differentiation of PC12K cells caused by depolarization with high KCl is mediated by endogenous NO. The outgrowth of neurites was significantly inhibited by 2 mM N-nitro-L-arginine methyl ester (L-NMAE), and 10 mM L-NAME was necessary for complete inhibition. The inhibition of NGF-dependent neurite outgrowth by L-NAME was abolished by depolarization of cells with KCl. The expression of neuronal- and endothelial-NO-synthase in PC12K cells was confirmed by immuno-cytochemical and immuno-blotting analyses with the respective monoclonal antibodies. However, the expression of inducible-NO synthase was not observed in PC12K cells cultured with high KCl under the depolarization conditions with 45 mM KCl. We observed the increase of NO in the differentiated PC12K cells using diaminofluorescein, a novel fluorescent indicator for NO.