N-CAM expression and localization in PC12 cells modulated by extracellular peptides

The neural cell adhesion molecules (N-CAMs) play an important role in mediating cell–cell interactions in the nervous system. Different isoforms of these membrane proteins are involved in the formation of the neuronal network and in the dynamic phases of neuronal plasticity.

We studied the early stages of the pseudo neuronal differentiation of PC12 cells induced by a class of small acidic peptides capable of modulating gene expression in these cells.

The data presented here indicate that peptides with specific sequences induce an increase in N-CAM mRNA expression and protein translocation to the plasma membrane to a comparable degree as NGF.     

Expanded CTG repeats inhibit neuronal differentiation of the PC12 cell line

Myotonic dystrophy (DM) is a dominant neuromuscular disorder caused by the expansion of trinucleotide CTG repeats in the 3-untranslated region (3'-UTR) of the MtPK gene. Although DM-associated mental retardation suggests that neuronal functions are disturbed by the expansion mutation, the effect of this alteration in neuronal cells has not been approached. In this study we established stable transfectans of PC12 neuronal cell line expressing the reporter gene CAT alone (empty-vector clone) or fused to the MtPK 3'-UTR with 5, 60, or 90 CTG repeats (CTG5, CTG60, and CTG90 clones, respectively). CTG90 cells exhibited a suppression of NGF-induced neuronal differentiation while empty-vector, CTG5 and CTG60 clones differentiated normally. CTG90 cells displayed normal activation of early differentiation markers, ERK1/2, but the up-regulation of the late marker MAP2 was dramatically reduced. Our neuronal cell system provides the first information of how the mutant MtPK 3'-UTR mRNA affects neuronal functions.     

Gi-coupled prostanoid receptors are the likely targets for COX-1-generated prostanoids in rat pheochromocytoma (PC12) cells

Cyclooxygenase-1 (COX-1) behaves as a delayed response gene in rat pheochromocytoma (PC12) cells exposed to nerve growth factor (NGF). To investigate the possible targets for COX-1 generated prostanoids in the early stages of neuronal differentiation, we have examined the expression of prostanoid receptors by PC12 cells using functional assays. Prostanoid receptor-specific agonists failed to activate adenylyl cyclase in undifferentiated and NGF-treated PC12 cells; neither did they stimulate phospholipase C activity. EP3 receptor agonists and PGF_2α were the only active ligands, able to inhibit forskolin-stimulated adenylyl cyclase activity. PC12 cells expressed EP3 and FP receptor mRNA, but only the responses to EP3 receptor agonists were inhibited by the EP3 receptor antagonist ONO-AE3-240. The functional role of NGF-stimulated COX-1 remains to be determined since we found no strong evidence of a role for EP3 receptors in the morphological changes induced by NGF during the early stages of differentiation of PC12 cells.     

IFN-gamma facilitates NGF-induced neuronal differentiation in PC12 cells

Natural or recombinant murine interferon-gamma causes a reversible arrest of proliferation of PC12 cells. Treatment with other antimitotics (AraC, colchicine, mitomycin C, hydroxyurea) or removal of serum, on the contrary, leads to mitotic arrest followed by cell death. IFN-gamma-treated PC12 cells respond more rapidly to NGF in terms of speed of neuronal outgrowth. On the other hand, NGF potentiates the action of IFN-gamma in stimulating the enzyme 2',5'-A synthetase which shifts from an average of 4.4-fold stimulation at 48 h with IFN-gamma alone to increments varying between 5- and 18-fold when PC12 cells are treated for 48 h with IFN-gamma and NGF. NGF alone, on the contrary, does not exert any detectable effect on this enzyme. From the findings we propose the use of a combined treatment of PC12 cells with NGF and IFN-gamma for a more rapid induction of neuronal differentiation.     

Two-dimensional surface plasmon resonance imager: An approach to study neuronal differentiation

There is a growing demand for the development of a new bioanalytical technique that is capable of monitoring neuronal differentiation noninvasively, in real time, and without any fluorescent probes. In a previous article, we demonstrated that a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager was very useful to monitor cell response on chemical stimulation in which protein kinase C (PKC) translocation was related. In the current study, we focused on developing a new method for monitoring neuronal differentiation and examined the application of the high-resolution 2D–SPR imager to monitor neuronal differentiation noninvasively and by a label-free format. We successfully monitored the intracellular signal transduction, which was mainly translocation of PKC in PC12 cells by the 2D–SPR imager, and found that the cells treated with a differentiation factor, nerve growth factor (NGF), showed a remarkable enhancement of 2D–SPR response to muscarine, carbachol, and acetylcholine stimulation. The results demonstrated that 2D–SPR sensing is applicable to in situ assessment of neuronal differentiation and to studying the expression state of the specific receptors in the living state.     

Ubiquitin and ubiquitin-protein conjugates in PC12h cells: Changes during neuronal differentiation

Ubiquitin and ubiquitin-protein conjugates in PC12h cells were detected with in vitro [¹²⁵I]ubiquitination, and quantified by immunoblotting. These levels were altered by nerve growth factor (NGF), which promotes neuronal differentiation. (i) Levels of high molecular weight (HMW) ubiquitin-protein conjugates ranging from 40 to 1,000kDa were increased by 2 days of NGF treatment, and remained high up to 10 days of NGF treatment. (ii) Ubiquitin and a 23-kDa conjugate tended to be decreased from days 2 to 10 of NGF treatment. 10-Day culture with 10 nM staurosporine, an protein kinase inhibitor, that blocks NGF-induced neurite outgrowth suppressed the NGF-induced increases in levels of HMW conjugates. Cyclic AMP and forskolin, both of which promote neurite outgrowth, mimicked the NGF-induced changes in ubiquitin and HMW conjugates, but phorbol ester and epidermal growth factor had little effect. These findings suggest that changes in ubiquitin-protein conjugates are closely coupled with neuronal differentiation.     

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.     

Differentiation of PC12 cells in three-dimensional collagen sponges with micropatterned nerve growth factor

Micropatterning of biological cues is important for the guided formation of neuronal outgrowth and neuronal differentiation. Nerve growth factor (NGF) was micropatterned in a three-dimensional collagen sponges by using micropatterned ice lines that were composed of collagen and NGF. The micropatterned ice lines were prepared by a dispersing machine. PC12 cells were cultured in the NGF-micropatterned collagen sponges and showed micropatterned neurite outgrowth. The neurite outgrowth followed the micropattern of NGF with more neurite outgrowth in the collagen/NGF lines than in the regions between the collagen/NGF lines. The micropattern of the NGF and the neurite network of the PC12 cells can be manipulated by controlling the micropattern of the NGF. The three-dimensional porous scaffolds prepared by this method will have a potential application for the regeneration and repair of the nervous system.     

Effects of vibration on differentiation of cultured PC12 cells

Different types of physiological‐mechanical stress, such as shear stress in vascular endothelial cells or hydrostatic pressure in chondrocytes are well known as regulators of cell function. In this study, the effects of vibration, a type of non‐physiological mechanical stimulation, on differentiation of rat pheochromocytoma (PC12) cells are reported. A nano‐vibration system was designed to produce nanometer‐scale vibration. The frequency and amplitude of the nano‐vibrations were monitored by a capacitance displacement sensor connected to an oscilloscope. When PC12 cells exposed to nerve growth factor were subjected to vibration at 10 kHz, differentiation and elongation of their neurites were promoted earlier in the culture. Vibration promoted differentiation of PC12 cells. This approach could therefore also be promising for determining of the effects of the physical environment on cell differentiation. Biotechnol. Bioeng. 2011; 108:592–599. © 2010 Wiley Periodicals, Inc.     

EGF-Induced sustained tyrosine phosphorylation and decreased rate of down-regulation of EGF receptor in PC12h-R cells which show neuronal differentiation in response to EGF

PC12h-R cell, a subclone of PC12 cells, exhibited a neuron-like phenotype, including neurite outgrowth and increased acetylcholinesterase activity, in response to epidermal growth factor (EGF) as well as nerve growth factor (NGF). We examined the mechanism by which EGF induced the neuronal differentiation in PC12h-R cells. The EGF-induced neuronal differentiation of PC12h-R cells was not blocked by K252a, whereas that induced by NGF was. EGF induced sustained tyrosine phosphorylation of the EGF receptor in PC12h-R cells, but not in the parent PC12h cells, which do not show neuronal differentiation in response to EGF. In addition, the rate of EGF-induced down-regulation of the EGF receptor in PC12h-R cells was decreased compared with that in PC12h cells. Furthermore, we found that the duration of EGF-induced tyrosine phosphorylation of the EGF receptor in PC12h-R cells was similar to that of NGF-induced tyrosine phosphorylation of p140 ^trkA in PC12h cells. The EGF-induced phosphorylation of the EGF receptor in PC12h cells was less sustained than that of p140 ^trkA by NGF in PC12h cells. These findings suggested that the EGF-induced neuronal differentiation of PC12h-R cells is due to the sustained activation of the EGF receptor, resulting from the decreased down-regulation of the EGF receptor and that the duration of the receptor tyrosine kinase activity determines the cellular responses of PC12 cells. We concluded that sustained activation of the receptor tyrosine kinase induces neuronal differentiation, although transient activation promotes proliferation of PC12 cells. Special issue dedicated to Dr. Hans Thoenen.     

Effect of chitooligosaccharide on neuronal differentiation of PC-12 cells

Chitosan is now being widely used biomaterial in the tissue engineering field, and has great potential as a candidate material for preparing nerve guidance conduits due to its various favorable properties, especially that of good nerve cell affinity. Chitosan can be degraded in vivo into chitooligosaccharide. We have investigated the in vitro effects of chitooligosaccharide on neuronal differentiation of PC-12 cells to see what effects chitooligosaccharide have on certain functions in the regenerating neurons. The morphologic observation and assessment using the specific reagent of tetrazolium salt WST-8 indicated that neurite outgrowths from PC-12 cells and the viability of PC-12 cells were enhanced by treatment of chitooligosaccharide. The real-time quantitative RT-PCR and Western blot analysis revealed showed that chitooligosaccharide could upregulate the expression of neurofilament-H mRNA or protein and N-cadherin protein in PC-12 cells. The maximum effect of 0.1 mg/ml chitooligosaccharide was obtained after 2 week culture. All the data suggest that chitooligosaccharide possesses good nerve cell affinity by supporting nerve cell adhesion and promoting neuronal differentiation and neurite outgrowth.     

Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells

Nitric oxide (NO) is a signaling molecule implicated in a spectrum of cellular processes including neuronal differentiation. The signaling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase and S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. However, little is known about the mechanisms involved and the target proteins for endogenous NO during the progression of neuronal differentiation. To address this question, we investigated the presence, localization, and subcellular distribution of nitrated proteins during neurotrophin-induced differentiation of PC12 cells. We find that some proteins show basal levels of tyrosine nitration in PC12 cells grown in the absence of nerve growth factor (NGF) and that nitration levels increase significantly after 2 days of incubation with this neurotrophin. Nitrated proteins accumulate over a period of several days in the presence of NGF. We demonstrate that this nitration is coupled to activation of nitric oxide synthase. The subcellular distribution of nitrated proteins changes during PC12 cell differentiation, displaying a shift from the cytosolic to the cytoskeletal fraction and we identified alpha-tubulin as the major target of nitration in PC12 cells by N-terminal sequence and MALDI-TOF analyses. We conclude that tyrosine nitration of proteins could be a novel molecular mechanism involved in the signaling pathway by which NO modulates NGF-induced differentiation in PC12 cells.     

NGF诱导PC12细胞分化的研究

动物实验表明,生理浓度的乙醇在脑发育过程中,不但可以影响神经细胞的数量,还可协同增强NGF诱导PC12细胞形态和功能上的分化,分化的PC12细胞具有与交感神经元相似的性状特征。用100mmol/L乙醇和50ng/mlNGF联合诱导可建立PC12细胞分化模型,为以神经细胞为研究对象的实验提供一种获得神经细胞的方法。     

An electroporation protocol for efficient DNA transfection in PC12 cells

A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.     

Binding-induced activation of overexpressed p185HER2 is essential in triggering neuronal differentiation of PC12 cells

To determine whether p185^HER2 overexpression per se triggers p185^HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185^HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185^HER2 MAbs, PC12–HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185^HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185^HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185^HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity. J. Cell. Biochem. 67:316–326, 1997. © 1997 Wiley-Liss, Inc.     

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.     

Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage

This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.