Inhibition of NGF-induced neurite outgrowth of rat pheochromocytoma cells (PC12) following administration of dioxyamphetamine

Amphetamine analogs are known to induce not only neurotoxicity at serotonergic axon terminals but also neocortical neuronal degeneration. However, a much less studied aspect involves the impact of amphetamine exposure on neuronal development. The present study investigated whether pretreatment of PC12 cells with dioxyamphetamine (DA) alters differentiation of PC12 cells by NGF and, if so, which components of the Ras/Raf/MEK/ERK pathway known to be involved in the differentiation response to NGF are particularly affected. Though exposure of PC12 cells to DA 1 h prior to NGF treatment resulted in apopotosis, several PC12 cells survived. However, neurite outgrowth of these NGF-responsive cells was repressed. Immunoblots of whole cell extracts revealed a strong induction rather than inhibition of ERK phosphorylation up to 48 h after DA/NGF treatment. Our results indicate that NGF-mediated neurite outgrowth was inhibited by pretreatment with DA, and this blockage of NGF-induced neuritogenesis was not due to an inhibition of ERK phosphorylation.     

ERK在NGF诱导PC12 细胞分化中的作用

目的探讨细胞外信号调节激酶(ERK)在NGF。诱导的PC12细胞分化中的作用机制。方法以NGF处理PC12细胞建立分化模型,运用免疫印迹检测不同浓度不同作用时间时NGF对ERK1／2蛋白和磷酸化ERK1／2蛋白水平的影响,并观察MAPK／ERK激酶(MEK)抑制剂U0126对NGF诱导的细胞形态学改变的影响。结果ERK1／2蛋白的磷酸化呈现NGF剂量和时间依赖性。NGF作用细胞5min即可观察到明显的ERK1／2蛋白磷酸化,持续1h左右,2h时降低到初始水平,而细胞形态的改变出现在NCF作用12h以后。倒置相差显微镜观察可见PC12细胞分化的程度与ERK1／2活化持续的时间正相关,U0126可完全即时抑制ERK1／2的活化,而ERK1／2活化的抑制可完全阻断。NGF诱导的PC12细胞分化。结论ERK1／2的活化是PC12细胞发生分化的必需事件,其活化时间的长短对分化具有决定作用。     

Neurite outgrowth of PC12 mutant cells induced by orange oil and d-limonene via the p38 MAPK pathway

We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.     

Functional role of JNK in neuritogenesis of PC12-N1 cells

JNKs, also known as SAPKs, are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. In the rat pheochromocytoma PC12 cells, the JNK signaling pathway mediates diverse functions such as differentiation and apoptosis. We have previously shown that activated JNK is required for later stages of neuritogeneis induced by NGF in a variant PC12 cell line (N1). Here, the functional role of JNK in N1 cells is further investigated. We show that NGF treatment, which induces extensive neurite branching and cell soma enlargement in the N1 cells, stimulates a biphasic activation of JNK. The first phase of activation is rapid and transient, beginning at 15 min after NGF exposure and lasting approximately 45 min. The second phase of activation is sustained, beginning at 9-12 h of NGF treatment and lasting for at least 24 h. Similar biphasic pattern of JNK activation is also observed in the parental PC12 cells. Using the specific JNK inhibitor SP600125, we show that the biphasic activation is necessary for neurite outgrowth and branching, and that inhibition of either phase suppresses neuritogenesis in the N1 cells. These results suggest that dynamic JNK activation may play a key role in neurite outgrowth during neuronal development.     

Dorsomorphin stimulates neurite outgrowth in PC12 cells via activation of a protein kinase A-dependent MEK-ERK1/2 signaling pathway

In this study, we investigated the effect of dorsomorphin, a selective inhibitor of bone morphogenetic protein (BMP) signaling, on rat PC12 pheochromocytoma cell differentiation. PC12 cells can be induced to differentiate into neuron-like cells possessing elongated neurites by nerve growth factor, BMP2, and other inducers. Cells were incubated with BMP2 and/or dorsomorphin, and the extent of neurite outgrowth was evaluated. Unexpectedly, BMP2-mediated neuritogenesis was not inhibited by co-treatment with dorsomorphin. We also found that treatment with dorsomorphin alone, but not another BMP signaling inhibitor, LDN-193189, induced neurite outgrowth in PC12 cells. To further understand the mechanism of action of dorsomorphin, the effects of this drug on intracellular signaling were investigated using the following signaling inhibitors: the ERK kinase (MEK) inhibitor U0126; the tropomyosin-related kinase A inhibitor GW441756; and the protein kinase A (PKA) inhibitor H89. Dorsomorphin induced rapid and sustained ERK1/2 activation; however, dorsomorphin-mediated ERK1/2 activation and neuritogenesis were robustly inhibited in the presence of U0126 or H89, but not GW441756. These findings suggest that dorsomorphin has the potential to induce neuritogenesis in PC12 cells, a response that requires the activation of PKA-dependent MEK-ERK1/2 signaling.     

Involvement of BREK, a serine/threonine kinase enriched in brain, in NGF signalling

We identified AATYK2 (Apoptosis-Associated Tyrosine Kinase 2) through a database search as a kinase specifically expressed in the brain. After characterization, we renamed it BREK (Brain-Enriched Kinase). Mouse BREK mRNA is expressed predominantly in brain, especially in olfactory bulb, olfactory tubercle, hippocampus, striatum, cerebellum, and cerebral cortex. Levels of expression and phosphorylation of BREK were high at 0-2 weeks after birth, suggesting that BREK is involved in neural development and functions during the early postnatal period. Phosphoamino acid analysis following in vitro kinase reaction revealed that BREK is a catalytically active, serine/threonine kinase. In PC12 cells, BREK was phosphorylated rapidly upon stimulation with nerve growth factor (NGF) in a protein kinase C-dependent pathway. In differentiated PC12 cells, BREK was enriched in cell bodies and growth cones, and also present along neurites. Introduction of a kinase-defective mutant of BREK into PC12 cells enhanced both ERK phosphorylation and neurite outgrowth in response to NGF, suggesting that BREK is a negative regulator of NGF-induced neuronal differentiation. Thus, we conclude that BREK is a new member of the family of protein serine/threonine kinases and that it plays important roles in NGF-TrkA signalling.     

Genistein enhances the NGF-induced neurite outgrowth

In the present report, we studied if an isoflavone, genistein, enhances the nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells. Application of genistein enhanced the NGF-induced neurite outgrowth. Knockdown of Na+/K+/2Cl- cotransporter isoform 1 (NKCC1) abolished the stimulatory effect of genistein on the neurite outgrowth. These observations indicate that NKCC1 is essential for genistein to stimulate the NGF-induced neurite outgrowth, although genistein had no effect on the protein expression of NKCC1. On the other hand, genistein activates NKCC1 as shown in our previous study. Taken together, these observations indicate that genistein enhanced the NGF-induced neurite outgrowth in PC12 cells via activation of NKCC1.     

Sustained ERK1/2 but not STAT1 or 3 activation is required for thanatophoric dysplasia phenotypes in PC12 cells

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause the most common genetic form of short-limbed dwarfism, achondroplasia (ACH), as well as neonatal lethal forms, thanatophoric dysplasia (TD) I and II. The causative mutations induce graded levels of constitutive activation of the receptor that correspond to the severity of the disorder, resulting in premature entry into hypertrophic differentiation and reduced proliferation of chondrocytes in developing cartilage. Although FGFR3 promotes growth in most tissues, it is a negative regulator of endochondral bone growth. Several signaling pathways have been implicated in these skeletal disorders including the Ras/MEK/ERK pathway and the JAK/STAT, the latter in the most severe phenotypes, however their functional relevance remains incompletely understood. Using PC12 cell lines stably expressing inducible mutant receptors containing the TDII mutation, K650E, sustained activation of ERK1/2 and activation of STAT1 and STAT3, but not STAT5, is observed in the absence of ligand. This activation leads to neurite outgrowth, a phenotypic readout of constitutive receptor activity, and sustained ERK1/2 activity is required for this ligand-independent differentiation. To assess the functional relevance of STAT activation induced by the mutant receptor, STATs were specifically downregulated using RNA-interference. Silencing of STAT1 or 3 independently or in combination had no significant effect on ligand-independent neurite outgrowth, ERK1/2 activation or p21(WAF1/CIP1) protein levels. These results support a model in which sustained activation of ERK1/2 is a key regulator of the increased transition to hypertrophic differentiation of the growth plate, whereas activation of STATs 1 and 3 is not required.     

Visfatin induces neurite outgrowth in PC12 cells via ERK1/2 signaling pathway

The angiogenic and inflammatory functions of visfatin and its effect on vascular cells, are fairly well known. However, its role within the nervous system remains largely unclear. To gain insight into this area, we studied the neuritogenic effect of visfatin on PC12 rat pheochromocytoma cells. We investigated whether visfatin gene expression, which is upregulated by hypoxia in cancer cells, is associated with neuritogenesis in PC12 cells. Using RT-PCR, Western blot analysis, ELISA, morphological observations, and immunostaining, we initially showed that CoCl₂, a hypoxic mimetic agent, upregulated visfatin gene expression along with neurite outgrowth in PC12 cells. We also showed that visfatin stimulated neurite outgrowth in PC12 cells. Moreover, in PC12 cells, visfatin evoked the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), which is closely linked to neuritogenesis. Visfatin-induced outgrowth of neurites was prevented by inhibition of the ERK1/2 pathway. Taken together, our results demonstrate for the first time that visfatin induces neurite outgrowth in PC12 cells via the activation of an ERK-dependent pathway, and suggest that visfatin may exert various biological, physiological, and pathological functions in not only the vascular system but also the nervous system.     

P53和P21在PC12细胞分化中的作用

目的探讨P53及其下游P21蛋白在PC12细胞分化中的可能作用机制。方法用反转录病毒质粒pBabe-P53/m175转染未分化PC12细胞,经筛选后建立野生型P53蛋白功能丧失的细胞系PC12(P53/m175);利用倒置相差显微镜、流式细胞术及Western blot等方法,比较两种细胞在神经生长因子(NGF)作用后,细胞分化表型、细胞周期及相关蛋白表达的改变。结果NGF作用于正常PC12细胞组,P53/P21蛋白表达持续升高,细胞G1期阻滞出现的时间与蛋白表达开始增加的时间相一致;而在NGF作用的PC12(P53/m175)细胞组,细胞不能表达P21蛋白,且细胞周期G1期阻滞的程度也出现显著下降。NGF作用下,两组细胞都能观察到神经突起的生长,表现出明显的分化表型。结论PC12细胞经NGF作用后出现P53及P21蛋白持续表达增加,主要介导了细胞分化过程中细胞周期G1期阻滞这一特征的出现,但并不是导致神经突起进行性生长这一特征的必需条件。     

The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells

The nervous system is highly sensitive to various environmental stresses, such as ischemia. Stress response mechanisms that result in neuroprotection, including the induction of heat shock proteins (HSP), are not well understood. We examined the effect of KNK437, a compound that inhibits the synthesis of inducible heat shock proteins, on neuronal differentiation in rat pheochromocytoma PC12 cells. KNK437 decreased the expression of HSP70, and induced the neurite outgrowth of PC12 cells in the absence of stress stimulation, although with lower efficacy than nerve growth factor (NGF). Neurite outgrowth stimulated by KNK437 and NGF was blocked by inhibitors of ERK mitogen-activated protein (MAP) kinase, p38 MAP kinase, and glycogen synthase kinase 3β signaling pathways. NGF, and not KNK437, induced acetylcholine esterase (AChE) activity, a functional differentiation marker, indicating that KNK437 utilizes a mechanism distinct from that of NGF. KNK437 enhanced the activity of low dose NGF treatment on neurite outgrowth induction and ERK phosphorylation in PC12 cells, a finding that identifies KNK437 as a possible nerve regeneration agent. This compound may be a useful tool for the investigation of neuronal differentiation and neuroprotection against environmental stress.     

Induction of neurite outgrowth in PC12 cells by the medium-chain fatty acid octanoic acid

It has been shown that polyunsaturated fatty acids such as arachinonic and docosahexanoic acids but not monounsaturated and saturated long-chain fatty acids promote basal and nerve growth factor (NGF)-induced neurite extension of PC12 cells, a line derived from a rat pheochromocytoma. On the other hand, short-chain fatty acids and valproic acid (2-propylpentanoic acid) enhance the growth of neurite processes of the cells only in the presence of inducers. In this study, we demonstrated that straight medium-chain fatty acids (MCFAs) at millimolar concentrations alone potently induced neuronal differentiation of PC12 cells. Hexanoic, heptanoic and octanoic acids dose-dependently induced neurite outgrowth of the cells: their maximal effects determined 2 days after addition to the culture medium were more marked than the effect of NGF. PC12 cells exposed to octanoic acid expressed increased levels of the neuronal marker beta-tubulin isotype III. Nonanoic, decanoic, and dodecanoic acids also induced growth of neurite processes, but their maximal effects were less marked than that of octanoic acid. In contrast, the polyunsaturated fatty acid linoleic acid and short-chain fatty acids had only slight or almost no effects on neurite formation in the absence of NGF. The effect of octanoic acid was synergistic with or additive to the effects of NGF and dibutyryl cyclic AMP. Octanoic acid upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), critical signaling molecules in neuronal differentiation, but not phosphorylation of Akt, a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K). Moreover, growth of neurites induced by octanoic acid was potently inhibited by treatment of cells with the p38 MAPK inhibitor SB203580 and the ERK kinase inhibitor PD98059 but not inhibited and only slightly inhibited by the JNK inhibitor SP600125 and the PI3K inhibitor wortmannin, respectively. Taken together, our results indicate that MCFAs, including octanoic acid, induced neurite outgrowth of PC12 cells in the absence of NGF and suggest that the activation of p38 MAPK and ERK pathways is involved in this process.     

Differences in neuritogenic response to nitric oxide in PC12 and PC12h cells

We have demonstrated that a natural iridoid compound, genipin, induces neurite outgrowth through the nitric oxide (NO)-cGMP-protein kinase G signaling pathway in PC12h cells. PC12 cells, the parental cell line of PC12h cells, have been shown to carry out neurite extension that accompanies NO production in response to nerve growth factor (NGF). This neurite outgrowth was significantly inhibited by NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, in both PC12 and PC12h cells, suggesting that the neuritogenesis is NO-dependent in both cells. In this report, we investigated whether genipin also induces neurite outgrowth in PC12 cells in order to determine the NO-dependent neurotrophic action of genipin in more than just one cell type. Genipin induced marked neurite outgrowth in PC12h cells but not in PC12 cells. The genipin-induced neurite outgrowth was significantly inhibited by L-NAME in PC12h cells. An NO donor, NOR4, also significantly induced neurite outgrowth in a concentration-dependent manner in PC12h cells but not in PC12 cells. On the other hand, NGF-primed PC12 cells exhibited significant neurite extension, which was inhibited by L-NAME, in response to genipin. Interestingly, NGF-primed PC12 cells responded to NOR4 extending neurites and expressed detectable neuronal NO synthase protein which is not detected in naive PC12 cells. These results suggest that genipin exerts a neuritogenic action on neuronal cells which are responsive to NO itself. Furthermore, the results also suggest that PC12h cells are more suitable for the study of NO-dependent neuronal function than PC12 cells which were not responsive to NO.     

Hypoxia induces neurite outgrowth in PC12 cells that is mediated through adenosine A2A receptors

Development of the nervous system is a complex process, involving coordinated regulation of diverse cellular processes including proliferation, differentiation and synaptogenesis. Disturbances to brain development such as pre- and perinatal hypoxia have been linked to behavioural and late onset of neurological disorders. This study examines the effect of hypoxia on neurite outgrowth in PC12 cells. Hypoxia not only caused a rapid induction of neurite outgrowth, but also synergistically enhanced nerve growth factor (NGF)-induced neurite outgrowth up to 24 h. Transactivation of TrkA receptors was ruled out since the TrkA inhibitor K252a did not block hypoxia-induced neurite outgrowth. Adenosine deaminase prevented hypoxia-induced neurite outgrowth indicating that the effect is mediated by adenosine. Use of the specific adenosine A2A receptor agonist CGS21680 and antagonist 8-3(chlorostyryl)caffeine demonstrated that activation of this receptor is critical for hypoxia-induced neurite outgrowth. Hypoxia-induced neurite outgrowth was blocked by the adenylate cyclase inhibitor, MDL-12,330A, indicating a role for activation of this enzyme in the pathway. Hypoxia was further shown to cause a decrease in growth-associated protein (GAP)-43 levels and a lack of induction of betaIII tubulin, in contrast to NGF treatment which resulted in increased cellular levels of both of these proteins. These findings suggest that hypoxia induces neurite outgrowth in PC12 cells via a pathway distinct from that activated by NGF. Thus, exposure to hypoxia at critical stages of development may contribute to aberrant neurite outgrowth and could be a factor in the pathogenesis of certain delayed developmental neurological disorders.     

Ganglioside GM1 binding toxins and human neuropathy-associated IgM antibodies differentially promote neuritogenesis in a PC12 assay

PC12 cells undergo neuritogenesis upon nerve growth factor (NGF) activation of the TrkA receptor, an effect mimicked by the ganglioside GM1 binding B-subunit of cholera toxin (CTB). Modulation of neuritogenesis by a GM1 ligand indicates a possible pathway for pathophysiological actions of neuropathy-associated anti-GM1 antibodies. Here we examine the ability of GM1 binding toxins and antibodies to induce neuritogenesis, using a PC12 neurite outgrowth assay. Cholera toxin (CT) and commercially prepared CTB (sCTB, contaminated with traces of the adenyl cyclase activating CT A-subunit) were highly neuritogenic. Recombinant cholera toxin B-subunit (rCTB, free from CTA) induced a much smaller effect, suggesting that the potent effects of sCTB are largely due to contaminating CTA. The recombinant GM1 binding B-subunit of Escherichia coli heat-labile enterotoxin (rETxB) exhibited no neuritogenic activity, whilst rETx holotoxin, which activates adenyl cyclase, was highly neuritogenic. Monoclonal anti-GM1 IgM antibodies from human neuropathy subjects induced small neuritogenic effects. These data indicate that GM1/ligand interaction does not necessarily lead to neuritogenesis and suggest that a specialisation of CTB, not shared by anti-GM1 antibodies or rETxB, is required to activate TrkA. Our data also indicate that antibodies are unlikely to exert major modulatory effects on TrkA activity in patients with anti-GM1 antibody-associated peripheral neuropathies.     

Involvement of P2Y13 receptor in suppression of neuronal differentiation

We examined the receptor-mediated effects of extracellular ATP on neuronal differentiation of PC12 cells, Neuro2a cells and MEB5 cells by using a series of receptor antagonists. The P2Y13 receptor antagonist MRS2211 significantly accelerated neurite outgrowth in all cases. Treatment with nerve growth factor (NGF) alone activated ERK1/2 in PC12 cells, and the activation was further increased by MRS2211. These results suggest involvement of P2Y13 receptor in suppression of neuronal differentiation. Thus, P2Y13 receptor antagonists might be candidates for treatment of neurodegenerative diseases.     

Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells

We previously found that the methanol extract of a marine brown alga, Sargassum macrocarpum showed marked nerve growth factor (NGF)-dependent neurite outgrowth promoting activity to PC12D cells. The active substance purified was elucidated to be sargachromenol. The median effective dose (ED50) was 9 microM against PC12D cells in the presence of 10 ng/ml NGF, although it showed no neurotrophic effect on its own. Pretreatment of cells with protein kinase A (PKA) inhibitor or U0126 substantially suppressed the sargachromenol-enhanced neurite outgrowth from PC12D cells, suggesting that the activation of cyclic AMP-mediated protein kinase and mitogen-activated protein (MAP) kinase 1/2 was apparently required for the action of sargachromenol. On the other hand, sargachromenol significantly promoted the survival of neuronal PC12D cells at 0-50 ng/ml NGF in serum-free medium. Neither PKA inhibitor nor U0126 could inhibit the survival supporting effect of sargachromenol, whereas wortmannin significantly blocked the sargachromenol-induced survival supporting effect on neuronal PC12D cells, suggesting that sargachromenol rescued neuronal PC12D cells by activating phosphatidylinositol-3 kinase. These results demonstrate that sargachromenol promotes neuronal differentiation of PC12D cells and supports the survival of neuronal PC12D cells via two distinct signaling pathways.     

Effect of scoparone on neurite outgrowth in PC12 cells

The neurite outgrowth-promoting effects of scoparone isolated from the stem bark of Liriodendron tulipifera were investigated in PC12 cells. At a concentration of 200 microM, scoparone markedly induced neurite outgrowth from PC12 cells. Scoparone at 200 microM also enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentration of Ca2+ were also increased by 200 microM scoparone. In addition, scoparone at 200 microM increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+/calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparone-induced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells.