Neuronal Differentiation Of PC12 Cells Research Articles

Up-regulation of Idh3α causes reduction of neuronal differentiation in PC12 cells

Up-regulation of Idh3α causes reduction of neuronal differentiation in PC12 cells

Up-regulation of Idh3α causes reduction of neuronal differentiation in PC12 cells

The PC12 is the widely used cell line to study neuronal differentiation. We had extensively investigated the details of protein expression in differentiated PC12 cells by proteomic analysis. The cells were incubated at the presence of nerve growth factor. We had analyzed the expression changes in the differentiating PC12 cells by 2-dimensional electrophoresis and the identification of the proteins using MALDI-TOF MS. By comparing expression pattern in the time course, we identified the candidate genes which are associated with neuronal differentiation. Among these genes, we performed real-time PCR analysis to validate Idh3alpha expression by the time course. To identify the function of Idh3alpha in neuronal differentiation stage, the transfection of Idh3alpha to PC12 cells was performed. As a result, we proved that up-regulation of Idh3alpha causes reduction in neural differentiation of PC12 cells. Based on these data, we suggest that Idh3alpha plays a role to the neuronal differentiation.

Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function

BackgroundAkt regulates various cellular processes, including cell growth, survival, and metabolism. Recently, Akt's role in neurite outgrowth has also emerged. We thus aimed to identify neuronal function-related genes that are regulated by Akt.MethodsWe performed suppression subtractive hybridization on two previously established PC12 sublines, one of which overexpresses the wild-type (WT) form and the other, the dominant-negative (DN) form of Akt. These sublines respond differently to NGF's neuronal differentiation effect.ResultsA variety of genes was identified and could be classified into several functional groups, one of which was developmental processes. Two genes involved in neuronal differentiation and function were found in this group. v-Maf musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) induces the neuronal differentiation of PC12 cells and immature telencephalon neurons, and synaptotagmin I (SytI) is essential for neurotransmitter release. Another gene, syntenin-1 (Syn-1) was also recognized in the same functional group into which MafK and SytI were classified. Syn-1 has been reported to promote the formation of membrane varicosities in neurons. Quantitative reverse transcription polymerase chain reaction analyses show that the transcript levels of these three genes were lower in PC12 (WT-Akt) cells than in parental PC12 and PC12 (DN-Akt) cells. Furthermore, treatment of PC12 (WT-Akt) cells with an Akt inhibitor resulted in the increase of the expression of these genes and the improvement of neurite outgrowth. These results indicate that dominant-negative or pharmacological inhibition of Akt increases the expression of MafK, SytI, and Syn-1 genes. Using lentiviral shRNA to knock down endogenous Syn-1 expression, we demonstrated that Syn-1 promotes an increase in the numbers of neurites and branches.ConclusionsTaken together, these results indicate that Akt negatively regulates the expression of MafK, SytI, and Syn-1 genes that all participate in regulating neuronal integrity in some way or another.

Role of STAT3 in In Vitro Transformation Triggered by TRK Oncogenes

TRK oncoproteins are chimeric versions of the NTRK1/NGF receptor and display constitutive tyrosine kinase activity leading to transformation of NIH3T3 cells and neuronal differentiation of PC12 cells. Signal Transducer and Activator of Transcription (STAT) 3 is activated in response to cytokines and growth factors and it has been recently identified as a novel signal transducer for TrkA, mediating the functions of NGF in nervous system. In this paper we have investigated STAT3 involvement in signalling induced by TRK oncogenes. We showed that TRK oncogenes trigger STAT3 phosphorylation both on Y705 and S727 residues and STAT3 transcriptional activity. MAPK pathway was involved in the induction of STAT3 phosphorylation. Interestingly, we have shown reduced STAT3 protein level in NIH3T3 transformed foci expressing TRK oncogenes. Overall, we have unveiled a dual role for STAT3 in TRK oncogenes-induced NIH3T3 transformation: i) decreased STAT3 protein levels, driven by TRK oncoproteins activity, are associated to morphological transformation; ii) residual STAT3 transcriptional activity is required for cell growth.

Neuronal adhesion and differentiation driven by nanoscale surface free-energy gradients

Recent results indicate that, in addition to chemical, spatial and mechanical cues, substrate physical cues such as gradients in surface energy may also impact cell functions, such as neuronal differentiation of PC12 cells. However, it remains to be determined what surface effect is the most critical in triggering PC12 cell differentiation. Here we show that, beyond continuously probing the surface energy landscape of their environment, PC12 cells are highly sensitive to nanoscale chemical heterogeneities. Self-assembled monolayers of alkylsiloxanes on glass were used as a culture substrate. By changing the structure, ordering and chemical nature of the monolayer, the surface energy distribution is altered. While both well-ordered CH 3 terminated substrates and bare glass (OH terminated) substrates did not favor PC12 cell adhesion, PC12 cells seeded on highly disordered CH 3/OH substrates underwent enhanced adhesion and prompt neuritogenesis by 48 h of culture, without nerve growth factor treatment. These data illustrate that surface free-energy gradients, generated by nanoscale chemical heterogeneities, are critical to biological processes such as nerve regeneration on biomaterials.

NRAGE is a negative regulator of nerve growth factor‐stimulated neurite outgrowth in PC12 cells mediated through TrkA‐ERK signaling

NRAGE, also denominated as MAGE-D1 or Dlxin-1, is firstly identified as a molecule interacting with NGF low affinity receptor p75NTR. It facilitates cell cycle arrest and NGF-dependent neuronal apoptosis. Here we report that NRAGE is downregulated while p75NTR is upregulated during the process of NGF-induced neuronal differentiation of PC12 cells. Knockdown of NRAGE by RNA interference accelerates NGF-mediated neurite outgrowth. In addition, in the NRAGE-suppressed cells, NGF-induced ERK activation is increased and this activation is MEK-dependent. Conversely, NRAGE overexpression significantly represses NGF-induced ERK activation. Further studies revealed that NRAGE downregulates TrkA expression through a post-transcriptional manner and thereby blocks NGF-induced TrkA phosphrylation at tyrosine-490. Altogether, these data indicate for the first time that NRAGE is an endogenous inhibitor for NGF-induced neuronal differentiation of PC12 cells by regulating TrkA-ERK signaling.

Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cells

BackgroundGene regulation at transcript level can provide a good indication of the complex signaling mechanisms underlying physiological and pathological processes. Transcriptomic methods such as microarray and quantitative real-time PCR require stable reference genes for accurate normalization of gene expression. Some but not all studies have shown that housekeeping genes (HGKs), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are routinely used for normalization, may vary significantly depending on the cell/tissue type and experimental conditions. It is currently unclear if these genes are stably expressed in cells undergoing drastic morphological changes during neuronal differentiation. Recent meta-analysis of microarray datasets showed that some but not all of the ribosomal protein genes are stably expressed. To test the hypothesis that some ribosomal protein genes can serve as reference genes for neuronal differentiation, a genome-wide analysis was performed and putative reference genes were identified based on stability of expressions. The stabilities of these potential reference genes were then analyzed by reverse transcription quantitative real-time PCR in six differentiation conditions.ResultsTwenty stably expressed genes, including thirteen ribosomal protein genes, were selected from microarray analysis of the gene expression profiles of GDNF and NGF induced differentiation of PC12 cells. The expression levels of these candidate genes as well as ACTB and GAPDH were further analyzed by reverse transcription quantitative real-time PCR in PC12 cells differentiated with a variety of stimuli including NGF, GDNF, Forskolin, KCl and ROCK inhibitor, Y27632. The performances of these candidate genes as stable reference genes were evaluated with two independent statistical approaches, geNorm and NormFinder.ConclusionsThe ribosomal protein genes, RPL19 and RPL29, were identified as suitable reference genes during neuronal differentiation of PC12 cells, regardless of the type of differentiation conditions. The combination of these two novel reference genes, but not the commonly used HKG, GAPDH, allows robust and accurate normalization of differentially expressed genes during PC12 differentiation.

CD38/cADPR/Ca2+ Pathway Promotes Cell Proliferation and Delays Nerve Growth Factor-induced Differentiation in PC12 Cells

Intracellular Ca(2+) mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca(2+) changes. Here we explored the role of one endogenous Ca(2+)-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca(2+) release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca(2+) increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.

Ligand-Dependent Activation of the Chimeric Tumor Necrosis Factor Receptor-Amyloid Precursor Protein (APP) Reveals Increased APP Processing and Suppressed Neuronal Differentiation

The β-amyloid precursor protein (APP) is a type I transmembrane protein whose functions and metabolic processing have been implicated in the pathogenesis of Alzheimer’s disease (AD). APP’s physical resemblance as a glycosylated receptor and the presence of several conserved motifs which are characteristics of a membrane-associated receptor has prompted interest to study and understand the role of downstream signaling events mediated by the activation of APP during both physiological and pathological conditions. Efforts to elucidate the mechanisms of APP signaling have not been conclusive. In order to further characterize the intracellular signaling activities of APP, we have constructed a chimeric APP receptor which is made up of the extracellular domain of human tumor necrosis factor receptor superfamily, member 1B (TNFRSF1B), the membrane-spanning segment of human APP₇₇₀ isoform’s β-cleaved carboxyl-terminal fragment (C99), and the green fluorescent protein (GFP) tagged to the carboxyl-terminus of C99. The mammalian rat pheochromocytoma (PC12) cell line is used as the cellular model for transfection of this <under>T</under>NFRSF1B-<under>A</under>PP-G<under>F</under>P (TAF) fusion receptor. Ligand-specific stimulation of this TAF receptor using human recombinant TNF reveals the induction of cytoplasmic TAF phosphorylation on Thr743 (numbering for APP₇₇₀ isoform) and an elevated release of APP intracellular domains (AICDs). Time-lapse microscopy shows the trafficking of GFP-tagged AICDs into the nucleus upon TAF receptor activation. The increased presence of AICDs is believed to suppress neuronal differentiation of PC12 cells in response to nerve growth factor treatment, by negatively regulating the levels of p53, cyclin D1 and phosphorylation of the signal transducer and activator of transcription STAT3.

Mechanisms involved in suppression of NGF-induced neuronal differentiation of PC12 cells by hyaluronic acid

In the present study, we found that hyaluronic acid (HA) suppressed the neuronal differentiation mediated by nerve growth factor (NGF). In addition, we examined the mechanism by which HA inhibits the NGF-induced neurite outgrowth of PC12 cells. We elucidated the direct interaction between NGF and HA, and found that HA did not bind to NGF directly using a quartz-crystal microbalance. Western blot analysis revealed that HA suppressed NGF-induced phosphorylation of p38 MAPK, ERKs, and transcriptional factor CREB in PC12 cells. Furthermore, HA inhibited the luciferase activity of pCRE-Luc transfected PC12 cells in the presence of NGF. We confirmed that the p38 MAPK inhibitor SB203580 and ERK inhibitor U0126 suppressed NGF-induced neurite outgrowth of PC12 cells, and found that the inhibitory effects of HA on phosphorylation of ERKs, but not of p38 MAPK, were restored by the anti-RHAMM antibody. The number of PC12 cells with neurites increased remarkably when pre-cultured with the anti-RHAMM antibody, then treated with NGF and HA. Our findings indicate that HA inhibits NGF-induced neuronal differentiation of PC12 cells partially by inhibiting ERK phosphorylation through RHAMM, and suggest that the binding of HA to RHAMM modifies the signaling pathways in PC12 cells treated with NGF.

Metabolites of sesamin, a major lignan in sesame seeds, induce neuronal differentiation in PC12 cells through activation of ERK1/2 signaling pathway

Sesamin, a major lignan in sesame seeds, exhibits various health benefits. Here, we investigated effects of sesamin, its stereoisomer episesamin, and their metabolites on neuronal differentiation in rat pheochromocytoma PC12 cells. Among all compounds tested, primary metabolites of sesamin and episesamin, SC-1 and EC-1 {S- and R-epimer of 2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0]octane}, were the most potent to induce neuronal differentiation. SC-1 alone induced neuronal differentiation through extracellular signal-regulated kinase (ERK) 1/2 activation that is essential for nerve growth factor (NGF)-induced neuronal differentiation, as shown by the suppression with MEK1/2 inhibitors, PD98059 and U0126. However, SC-1 did not increase phosphorylation of TrkA, a high-affinity NGF receptor, and a TrkA inhibitor, K252a, did not affect SC-1-induced neuronal differentiation. Furthermore, SC-1 potentiated neuronal differentiation in cells co-treated with NGF, which was associated with enhanced ERK1/2 activation and increased expression of neuronal differentiation markers. Interestingly, when treated with SC-1 and a high dose of NGF, formation of synaptic connections and synaptophysin accumulation at the neurite terminals were markedly enhanced. These results indicate that (1) SC-1 alone induces neuronal differentiation, (2) SC-1 potentiates neuronal differentiation in NGF-treated cells, (3) SC-1 enhances formation of synaptic connections in cells treated with a high dose of NGF, all of which are associated with ERK1/2 activation. It is therefore concluded that SC-1 may promote neuronal differentiation by tapping into the ERK1/2-MAPK (mitogen-activated protein kinase) signaling pathway downstream from the TrkA receptor in PC12 cells.

Regulator of G-Protein Signaling 14 (RGS14) Is a Selective H-Ras Effector

BackgroundRegulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Gα-mediated GTP hydrolysis (“GTPase-accelerating proteins” or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Gα GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras.Methodology/Principal FindingsFull-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling.Conclusions/SignificanceIn cells, RGS14 facilitates the formation of a selective Ras·GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.

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.

Calcium overload is essential for the acceleration of staurosporine-induced cell death following neuronal differentiation in PC12 cells

Differentiation of neuronal cells has been shown to accelerate stress-induced cell death, but the underlying mechanisms are not completely understood. Here, we find that early and sustained increase in cytosolic ([Ca2(+)]c) and mitochondrial Ca2(+) levels ([Ca2(+)]m) is essential for the increased sensitivity to staurosporine- induced cell death following neuronal differentiation in PC12 cells. Consistently, pretreatment of differentiated PC12 cells with the intracellular Ca2(+)-chelator EGTA-AM diminished staurosporine-induced PARP cleavage and cell death. Furthermore, Ca2(+) overload and enhanced vulnerability to staurosporine in differentiated cells were prevented by Bcl-XL overexpression. Our data reveal a new regulatory role for differentiation-dependent alteration of Ca2(+) signaling in cell death in response to staurosporine.

Neuronal Differentiation of PC12 Cells Cultured on Growth Factor-Loaded Nanoparticles Coated on PLGA Microspheres

The development of nanotechnology has penetrated the fields of biology and medicine, resulting in remarkable applications for tissue regeneration. In order to apply this technology to tissue engineering, we have developed nanoscaled 3D scaffolds consisting of growth factor-loaded heparin/poly(l-lysine) nanoparticles (NPs) attached to the surface of polymeric microspheres via polyionic complex methods. Growth factor-loaded NPs were simply produced as polyelectrolyte complexes with diameters of 100-200 nm. They were then coated onto positively charged poly(lacticco- glycolic acid) (PLGA) pretreated with polyethyleneimine to enable cell adhesion, proliferation, and stimulation of neurite outgrowth. Propidium iodide staining and beta-tubulin analysis revealed that neuronal PC12 cells proliferated extensively, expressed significant amounts of b-tubulin, and showed well-structured neurite outgrowth on polymeric microspheres by stimulation with growth factors. These results suggest that cellular adhesion and biological functionality on prepared PLGA microspheres enabled terminal differentiation of neuronal cells.

Differential and Synergistic Effect of Nerve Growth Factor and cAMP on the Regulation of Early Response Genes during Neuronal Differentiation

Neurotrophin (NT)-driven differentiation is a process involving activation of multiple signalling events. Treatment of PC12 cells with the prototypic NT nerve growth factor (NGF) induces PC12 cell differentiation characterized by neurite outgrowth and expression of differentiation genes. Cyclic AMP (cAMP), one of the second messengers of NGF stimulation, has also been observed to induce neuronal differentiation in PC12 cells. Interestingly, co-treatment of NGF and dibutyryl cAMP (DBcAMP) exhibits a synergistic effect on neurite outgrowth in PC12 cells, but the mechanisms underlying this synergism remain unknown. In the current study, we compared the gene expression profiles of PC12 cells treated with NGF, DBcAMP or both for 12 h to identify differentially regulated genes during the early stage of differentiation. We found that the genes that were differentially regulated by NGF, DBcAMP or both include genes for acquiring neuronal phenotypes, cytoskeleton-binding proteins and cell cycle proteins. Importantly, we identified a subset of genes that was specifically regulated during co-treatment of NGF and cAMP, suggesting that the synergistic effect of NGF and DBcAMP on neurite outgrowth is possibly mediated through transcription regulation. Our observations provide novel insights on the signalling mechanisms underlying the regulation of neuronal differentiation by NGF and cAMP.

The role of integrative nuclear fgfr1 signaling (infs) in pc12 cell neuronal differentiation

The role of integrative nuclear fgfr1 signaling (infs) in pc12 cell neuronal differentiation

Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in the center of the nucleoskeletal structure. In summary , we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.

Expression profile identifies novel genes involved in neuronal differentiation

In the presence of NGF, PC12 cells extend neuronal processes, cease cell division, become electrically excitable, and undergo several biochemical changes that are detectable in developing sympathetic neurons. We investigated the expression pattern of the apoptosis-related genes at each stage of neuronal differentiation using a cDNA microarray containing 320 apoptosis-related rat genes. By comparing the expression patterns through time-series analysis, we identified candidate genes that appear to regulate neuronal differentiation. Among the candidate genes, HO2 was selected by real-time PCR and Western blot analysis. To identify the roles of selected genes in the stages of neuronal differentiation, transfection of HO2 siRNA in PC12 cells was performed. Down-regulation of HO2 expression causes a reduction in neuronal differentiation in PC12 cells. Our results suggest that the HO2 gene could be related to the regulation of neuronal differentiation levels.

Sialic acid metabolism is involved in the regulation of gene expression during neuronal differentiation of PC12 cells

Sialic acid precursors are mediators of the sialic acid pathway. In this manuscript we present evidence that the application of sialic acid a precursor modulates gene expression and cell differentiation. The concept that sugars are involved in cellular transcription was first proposed by Jacob and Monod nearly 40 years ago studying the regulation of the lac-operon in prokaryotes. Surprisingly, these findings have never been transferred to eukaryotic systems. For our studies we have chosen PC12 cells. PC12-cells differentiate after application of NGF into a neuron-like phenotype. It is shown that treatment of PC12 cells with two different sialic acid precursors N-acetyl- or N-propanoylmannosamine, without application of NGF also induces neurite outgrowth. Moreover, the PC12 cells show the same morphology as the NGF-treated cells. Surprisingly, after application of both sialic acid precursors the phosphorylation and translocation of erk1/2 into the nucleus are activated, thus influencing the expression of genes involved in the differentiation of cells, such as the transcription factor c-Jun or TOAD-64/Ulip/CRMP (Turned ON After Division, 64 kd/ unc-33-like phosphoprotein/Collapsin Response Mediator Protein). These are the first experimental data showing that the sialic acid metabolism is closely associated with signal transduction and regulation of neuronal differentiation.