Neurite Formation In PC12 Cells Research Articles

Cell guidance on peptide micropatterned silk fibroin scaffolds

Guiding neuronal cell growth is desirable for neural tissue engineering but is very challenging. In this work, a self-assembling ultra-short surfactant-like peptide I3K which possesses positively charged lysine head groups, and hydrophobic isoleucine tails, was chosen to investigate its potential for guiding neuronal cell growth. The peptides were able to self-assemble into nanofibrous structures and interact strongly with silk fibroin (SF) scaffolds, providing a niche for neural cell attachment and proliferation. SF is an excellent biomaterial for tissue engineering. However neuronal cells, such as rat PC12 cells, showed poor attachment on pure regenerated SF (RSF) scaffold surfaces. Patterning of I3K peptide nanofibers on RSF surfaces significantly improved cellular attachment, cellular density, as well as morphology of PC12 cells. The live / dead assay confirmed that RSF and I3K have negligible cytotoxicity against PC12 cells. Atomic force microscopy (AFM) was used to image the topography and neurite formation of PC12 cells, where results revealed that self-assembled I3K nanofibers can support the formation of PC12 cell neurites. Immunolabelling also demonstrated that coating of I3K nanofibers onto the RSF surfaces not only increased the percentage of cells bearing neurites but also increased the average maximum neurite length. Therefore, the peptide I3K could be used as an alternative to poly-l-lysine for cell culture and tissue engineering applications. As micro-patterning of neural cells to guide neurite growth is important for developing nerve tissue engineering scaffolds, inkjet printing was used to pattern self-assembled I3K peptide nanofibers on RSF surfaces for directional control of PC12 cell growth. The results demonstrated that inkjet-printed peptide micro-patterns can effectively guide the cell alignment and organization on RSF scaffold surfaces, providing great potential for nerve regeneration applications.

Micropatterned nanolayers immobilized with nerve growth factor for neurite formation of PC12 cells.

BackgroundNerve regeneration is important for the treatment of degenerative diseases and neurons injured by accidents. Nerve growth factor (NGF) has been previously conjugated to materials for promotion of neurogenesis.Materials and methodsPhotoreactive gelatin was prepared by chemical coupling of gelatin with azidobenzoic acid (P-gel), and then NGF was immobilized on substrates in the presence or absence of micropatterned photomasks. UV irradiation induced crosslinking reactions of P-gel with itself, NGF, and the plate for immobilization.ResultsBy adjustment of the P-gel concentration, the nanometer-order height of micropatterns was controlled. NGF was quantitatively immobilized with increasing amounts of P-gel. Immobilized NGF induced neurite outgrowth of PC12 cells, a cell line derived from a pheochromocytoma of the rat adrenal medulla, at the same level as soluble NGF. The immobilized NGF showed higher thermal stability than the soluble NGF and was repeatedly used without loss of biological activity. The 3D structure (height of the formed micropattern) regulated the behavior of neurite guidance. As a result, the orientation of neurites was regulated by the stripe pattern width.ConclusionThe micropattern-immobilized NGF nanolayer biochemically and topologically regulated neurite formation.

Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling.

Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5’-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins.

Blocking Nerve Growth Factor Signaling Reduces the Neural Invasion Potential of Pancreatic Cancer Cells.

Perineural invasion (PNI) is thought to be one of the factors responsible for the high rate of tumor recurrence after surgery and the pain generation associated with pancreatic cancer. Signaling via the nerve growth factor (NGF) pathway between pancreatic cancer cells and the surrounding nerves has been implicated in PNI, and increased levels of these proteins have been correlated to poor prognosis. In this study, we examine the molecular mechanism of the NGF signaling pathway in PNI in pancreatic cancer. We show that knocking down NGF or its receptors, TRKA and p75NTR, or treatment with GW441756, a TRKA kinase inhibitor, reduces the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, pancreatic cancer cells migrate towards dorsal root ganglia (DRG) in a co-culture assay, indicating a paracrine NGF signaling between the DRGs and pancreatic cancer cells. Knocking down the expression of NGF pathway proteins or inhibiting the activity of TRKA by GW441756 reduced the migratory ability of Mia PaCa2 towards the DRGs. Finally, blocking NGF signaling by NGF neutralizing antibodies or GW441756 inhibited the neurite formation in PC-12 cells in response to conditioned media from pancreatic cancer cells, indicating a reciprocal signaling pathway between the pancreatic cancer cells and nerves. Our results indicate that NGF signaling pathway provides a potential target for developing molecularly targeted therapies to decrease PNI and reduce pain generation. Since there are several TRKA antagonists currently in early clinical trials they could now be tested in the clinical situation of pancreatic cancer induced pain.

Critical role of the miR-200 family in regulating differentiation and proliferation of neurons.

The generation of differentiated and functional neurons is a complex process, which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200, miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells, induction of miR-200 family was observed in differentiating neural stem cells, demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However, inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells, an inverse relationship was observed between expression of reprogramming transcription factors (SOX2, KLF4, NANOG, OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover, we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion, our results indicate that increased expression of the miR-200 family promotes neuronal differentiation, while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4.

The histone deacetylase inhibitor trichostatin A induces neurite outgrowth in PC12 cells via the epigenetically regulated expression of the nur77 gene

Histone deacetylase (HDAC) inhibitors induce histone acetylation and gene expression by changing local chromatin structures. They can thereby influence various cells to proliferate or differentiate. It has been reported that trichostatin A (TSA) or valproic acid (VPA) can induce the neuronal differentiation of mouse embryonic neural stem cells and rat cerebellar granule cells. It is unclear however which gene is responsible for the neuronal differentiation induced by HDAC inhibitors. In this study, we investigated the contribution of immediate early gene (IEG) nur77 to the neuronal differentiation induced by TSA. We report that TSA induces neurite outgrowth in PC12 cells, and C646, an inhibitor of HAT (histone acetyl transferase) (p300), prevents TSA-induced neurite formation. The acetylation of the Lys14 residue of histone H3, and mRNA and protein expression of nur77 gene were found to be stimulated after treatment with TSA, but not in the presence of C646. A knock-down of nur77 inhibits the neurite outgrowth induced by TSA. Furthermore, the ectopic expression of nur77 significantly elicits neurite formation in PC12 cells. These results suggest that the expression of nur77, which is up-regulated via the TSA-induced acetylation of Lys14 on histone H3, is essential for the neuronal differentiation in TSA-induced PC12 cells.

613 微粒子構造上での細胞培養と神経突起形成に関する研究

613 微粒子構造上での細胞培養と神経突起形成に関する研究

Dimethylarginine dimethylaminohydrolase 1 regulates nerve growth factor‐promoted differentiation of PC12 cells in a nitric oxide‐dependent but asymmetric dimethylargenine‐independent manner

There are significant morphological and biochemical alterations during nerve growth factor (NGF)-promoted neuronal differentiation, and the process is regulated by molecules, including nitric oxide (NO). Dimethylarginine dimethylaminohydrolase (DDAH) is thought to play a critical role in regulating NO production via hydrolyzing the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Thus, we tested the role of DDAH in NGF-promoted differentiation of PC12 (pheochromocytoma) cells. The present results show that both mRNA and protein levels of DDAH1 were increased, whereas those of DDAH2 were decreased, during NGF-promoted cell differentiation. Both the DDAH activity and the ADMA level in cultured medium were unchanged in this process. NGF promoted neurite formation and induced the expression of microtubule-associated protein 2 (MAP2), a neuronal marker, which were both significantly repressed by DDAH1 silence with small interfering RNA but not by DDAH2 silence. The expressions of three isoforms of NOS were markedly upregulated after NGF stimulation with a time course similar to that of DDAH1, which were attenuated by DDAH1 silence. Conversely, overexpression of DDAH1 accelerated neurite formation in PC12 cells, concomitantly with upregulating the expression of three NOS isoforms. In summary, our data reveal the critical regulatory effect of DDAH1 on NGF-promoted differentiation of PC12 cells in an NOS/NO-dependent but ADMA-independent manner.

Neurite outgrowth from PC12 cells by basic fibroblast growth factor (bFGF) is mediated by RhoA inactivation through p190RhoGAP and ARAP3

The rat pheochromocytoma cell line PC12 has been widely used as a model to study neuronal differentiation. PC12 cells give rise to neurites in response to basic fibroblast growth factor (bFGF). However, it is unclear whether bFGF promotes neurite outgrowth by inducing RhoA inactivation, and a mechanism for RhoA inactivation in PC12 cells in response to bFGF has not been reported. Lysophosphatidic acid (LPA) treatment and the expression of constitutively active (CA)-RhoA (RhoA V14) impaired neurite formation in response to bFGF, while Tat-C3 exoenzyme and the expression of dominant negative (DN)-RhoA (RhoA N19) stimulated neurite outgrowth. GTP-bound RhoA levels were reduced in response to bFGF, which suggests that the inactivation of RhoA is essential to neurite outgrowth in response to bFGF. To investigate the mechanism of RhoA inactivation, this study examined the roles of p190RhoGAP and Rap-dependent RhoGAP (ARAP3). DN-p190RhoGAP prevented neurite outgrowth, while WT-p190RhoGAP and Src synergistically stimulated neurite outgrowth; these findings suggest that bFGF promotes the inactivation of RhoA and subsequent neurite outgrowth through p190RhoGAP and Src. Furthermore, DN-Rap1 and DN-ARAP3 reduced neurite formation in PC12 cells. These results suggest that RhoA is likely to be inactivated by p190RhoGAP and ARAP3 during neurite outgrowth in response to bFGF.

NGF-induced moesin phosphorylation is mediated by the PI3K, Rac1 and Akt and required for neurite formation in PC12 cells

Moesin is a member of ERM family proteins which act as the cross-linkers between plasma membrane and actin-cytoskeleton and is activated by phosphorylation at Thr-558. In neurons, suppression of radixin and moesin alters the growth cone morphology. However, the significance of phosphorylation of ERM proteins in neuronal cells has not been fully investigated. In this study, we studied the signaling pathways responsible for moesin phosphorylation and its functional importance in NGF-treated PC12 cells. NGF rapidly induced the phosphorylation of moesin at Thr-558 in PC12 cells which was dependent on PI3K and Rac1. We found that Akt interacted and phosphorylated with moesin both in vitro and in vivo. Inhibition of PI3K and Rac1 abolished the NGF-induced Akt activation, indicating that Akt is at the downstream of PI3K and Rac1. To examine the functional role of phosphorylated ERM proteins, a dominant negative mutant form of moesin (T558A) was introduced into PC12 cells. The mutant significantly reduced the frequency of cells with neurites following NGF treatment. Our results indicate that PI3K, Rac1 and Akt-dependent phosphorylation of moesin is required for the NGF-induced neurite formation in differentiating PC12 cells.

Characterization of the cell membrane-associated products of the Neuregulin 4 gene

The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.

Versatile Roles of R-Ras GAP in Neurite Formation of PC12 Cells and Embryonic Vascular Development

Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.

Homophilic interactions of chick neurofascin in trans are important for neurite induction

Neurofascin is a member of the immunoglobulin superfamily involved in axon extension and fasciculation. Here we apply adenoviral short hairpin RNA (shRNA) expression in primary neurons, PC12-NIH/3T3 co-cultures in combination with Luminex assays, to demonstrate homophilic interactions of neurofascin for neurite outgrowth. An adenoviral vector was constructed for the expression of shRNA in primary tectal cells that inhibits gene expression similar to short interfering RNA. We demonstrate that after shRNA-mediated knockdown neuronal neurofascin expression is important for neurite outgrowth on a neurofascin substrate. Neurite outgrowth assays reveal that neurite formation of PC12 cells is increased when neurofascin is overexpressed on both outgrowing PC12 cells and substrate NIH/3T3 cells, suggesting that neurofascin expression is also sufficient for neurite induction. Luminex technology for the analysis of protein-protein interactions showed homophilic binding of neurofascin to itself.

Suppression of MeCP2β expression inhibits neurite extension in PC12 cells

Regulation of gene expression is critical to the proper development of neuronal cells. The methyl-CpG binding protein 2 (MeCP2) operates as a transcriptional repressor by facilitating histone deacetylation and DNA methylation-dependent transcriptional silencing. This study examined the importance of MeCP2 in the regulation of neurite formation in PC12 cells. Expression of MeCP2 increased in a time-dependent manner after induction of neuronal differentiation. Expression was assessed at both the transcriptional and translation levels, and reached a maximum at 24 h post-induction. In addition, a marked inhibition of neurite extension and proper localization of a marker for synapse formation, synapsin I, were observed when MeCP2 expression was decreased by the addition of an antisense morpholino oligomer directed to the translational initiation site for MeCP2β. The removal of the antisense oligomer allowed neurite extension to progress. However, the addition of antisense oligomer to previously differentiated PC12 cells did not affect established neurite processes. Taken collectively, our results indicate a role for MeCP2β early in the events of neurite formation and that the relative levels of MeCP2α and MeCP2β may be different in early differentiating neurons than is found in the adult brain. In addition, unique functions may exist for the two isoforms of MeCP2. Our results indicate that the inhibition of neurite elaboration caused by a reduction in MeCP2 may be reversible.

The catalytic activity, but not receptor binding, of sPLA 2s plays a critical role for neurite outgrowth induction in PC12 cells

We previously showed that fungal secretory phospholipase A 2 (sPLA 2) induces neurite formation in PC12 cells in an L-type Ca 2+ channel activity-dependent manner. In this study we compared neurite-inducing activity of different sPLA 2s, including bee venom sPLA 2 (bvPLA 2), and found that it correlated with the ability of each sPLA 2 to release fatty acids from live PC12 cells. Consistently, using several mutants of bvPLA 2, we found that the enzymatic activity rather than the binding activity to the putative N-type receptor for neurotoxic sPLA 2s is the critical determinant for the neuritogenic response. These results imply that the neurite outgrowth is elicited by the messenger(s) produced upon degradation of membrane phospholipids.

The effect of surface wettability on induction and growth of neurites from the PC-12 cell on a polymer surface

Surface properties of polymeric devices that are used to regenerate nervous damage are a point to be considered for axon regeneration in nerve system. In our previous studies, we prepared a wettability gradient on polyethylene (PE) surfaces using a corona discharge treatment from a knife-type electrode whose power increases gradually along the sample length. The PE surfaces were oxidized gradually with increasing power. The effect of surface wettability on the different types of cells has an important role for cell adhesion and proliferation. The purpose of this study is to investigate neurite formation on polymer surfaces with different wettability. Induction and growth of neurites from the rat pheochromocytoma (PC-12) cells attached on the polymer surfaces with different hydrophilicity were investigated using the wettability gradient PE surfaces prepared by a corona discharge treatment. Neurites were investigated for number and length of neurites in terms of surface wettability. It was observed that neurite formation of PC-12 cells was increased more onto the positions with moderate hydrophilicity of the wettability gradient surface than onto the more hydrophobic or hydrophilic positions. From those results, it could be assumed that initial adhesion of PC-12 cells was caused by more calf serum (CS) protein than nerve growth factor (NGF), whereas the neurite formation of PC-12 cells was caused by more NGF than CS protein. It follows from what has been said thus far that PC-12 cells are a differentiated neuronal phenotype with a long neurite at around the position 2.5 cm (water contact angle of about 55 deg). In conclusion, surface wettability plays an important role for neurite formation on the polymer surfaces for axon regeneration.

A Raf-1 mutant that dissociates MEK/extracellular signal-regulated kinase activation from malignant transformation and differentiation but not proliferation.

It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.

Somatostatin enhances neurite outgrowth in PC12 cells

The rat pheochromocytoma cell line PC12 forms neurites in response to nerve growth factor (NGF), and it was also reported to extend processes in the presence of somatostatin (somatotropin release-inhibiting factor, SRIF), a neuroactive peptide that seems to act as a morphogenetic factor in the developing nervous system. In the present study, we re-evaluated the effects of SRIF on PC12 cell differentiation. Our results indicate that SRIF alone is ineffective in promoting neurite outgrowth. Instead, SRIF or its analogue, octreotide (a SRIF agonist on the receptor subtypes 2, 3 and 5), potentiates neurite extension induced by NGF. These results suggest that SRIF enhances neurite formation in PC12 cells without directly promoting neurite outgrowth. SRIF potentiation of NGF-induced neurite outgrowth persists at least in part in the presence of pertussis toxin (PTX), suggesting the involvement of PTX-insensitive G-proteins. In addition, protein kinase-dependent pathways are likely to mediate SRIF effects on NGF-induced differentiation.

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

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

Nerve growth factor induced differentiation of neuronal cells requires gene methylation.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of gene methylation during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor (NGF). Here we present evidence that NGF-induced neuronal differentiation is dependent on gene methylation and that this process is not associated with inhibition of cell cycle arrest. The DNA methylation inhibitor 5-azacytidine is able to block the neurite outgrowth of NGF-treated PC12 cells. Inhibition of neuronal differentiation is accompanied by significant changes in the protein and mRNA expression pattern of the high-affinity NGF receptor (trkA). These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation dependent on gene methylation. The results indicate that DNA methyltransferase is necessary for the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in growth factor-dependent cellular responses distinct from cell proliferation.