RA-induced Neural Differentiation Research Articles

Investigating neural differentiation of mouse P19 embryonic stem cells in a time-dependent manner by bioinformatic, microscopic and transcriptional analyses.

As an available cell line, mouse pluripotent P19 has been widely employed for neuronal differentiation studies. In this research, by applying the in vitro differentiation of this cell line into neuron-like cells through retinoic acid (RA) treatment, the roles of some genes including DNMT3B, ICAM1, IRX3, JAK2, LHX1, SOX9, TBX3 and THY1 in neural differentiation was investigated. Bioinformatics, microscopic, and transcriptional studies were conducted in a time-dependent manner after RA-induced neural differentiation. According to bioinformatics studies, we determined the engagement of the metabolic and developmental super-pathways and pathways in neural cell differentiation, particularly focusing on the considered genes. According to our qRT-PCR analyses, JAK2, SOX9, TBX3, LHX1 and IRX3 genes were found to be significantly overexpressed in a time-dependent manner (p < 0.05). In addition, the significant downregulation of THY1, DNMT3B and ICAM1 genes was observed during the experiment (p < 0.05). The optical microscopic investigation showed that the specialized extensions of the neuron-like cells were revealed on day 8 after RA treatment. Accordingly, the neural differentiation of P19 cell line and the role of the considered genes during the differentiation were proved. However, our results warrant further in vivo studies.

The deubiquitinating enzyme UCHL1 is a favorable prognostic marker in neuroblastoma as it promotes neuronal differentiation

BackgroundNeuroblastoma (NB) is the most common pediatric solid tumor that originates from neural crest-derived sympathoadrenal precursor cells that are committed to development of sympathetic nervous system. The well differentiated histological phenotype of NB tumor cells has been reportedly associated with favorable patient outcome. Retinoic acid (RA) can effectively induce NB cell differentiation, thereby being used in the clinic as a treatment agent for inducing the differentiation of high-risk NB. However, the underlying molecular mechanisms of regulating differentiation remain elusive.MethodsThe correlation between clinical characteristics, survival and the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) expression were assessed using a neuroblastic tumor tissue microarray, and then validated in three independent patient datasets. The different expression of UCHL1 in ganglioneuroblastoma, ganglioneuroma and NB was detected by immunohistochemistry, mass spectra and immunoblotting analysis, and the correlation between UCHL1 expression and the differentiated histology was analyzed, which was also validated in three independent patient datasets. Furthermore, the roles of UCHL1 in NB cell differentiation and proliferation and the underlying mechanisms were studied by using short hairpin RNA and its inhibitor LDN57444 in vitro.ResultsBased on our neuroblastic tumor tissue microarrays and three independent validation datasets (Oberthuer, Versteeg and Seeger), we identified that UCHL1 served as a prognostic marker for better clinical outcome in NB. We further demonstrated that high UCHL1 expression was associated with NB differentiation, indicated by higher UCHL1 expression in ganglioneuroblastomas/ganglioneuromas and well-differentiated NB than poorly differentiated NB, and the positive correlation between UCHL1 and differentiation markers. As expected, inhibiting UCHL1 by knockdown or LDN57444 could significantly inhibit RA-induced neural differentiation of NB tumor cells, characterized by decreased neurite outgrowth and neural differentiation markers. This effect of UCHL1 was associated with positively regulating RA-induced AKT and ERK1/2 signaling activation. What’s more, knockdown of UCHL1 conferred resistance to RA-induced growth arrest.ConclusionOur findings identify a pivotal role of UCHL1 in NB cell differentiation and as a prognostic marker for survival in patients with NB, potentially providing a novel therapeutic target for NB.

Epigenetic regulation of human SOX3 gene expression during early phases of neural differentiation of NT2/D1 cells.

Sox3/SOX3 is one of the earliest neural markers in vertebrates. Together with the Sox1/SOX1 and Sox2/SOX2 genes it is implicated in the regulation of stem cell identity. In the present study, we performed the first analysis of epigenetic mechanisms (DNA methylation and histone marks) involved in the regulation of the human SOX3 gene expression during RA-induced neural differentiation of NT2/D1 cells. We show that the promoter of the human SOX3 gene is extremely hypomethylated both in undifferentiated NT2/D1 cells and during the early phases of RA-induced neural differentiation. By employing chromatin immunoprecipitation, we analyze several histone modifications across different regions of the SOX3 gene and their dynamics following initiation of differentiation. In the same timeframe we investigate profiles of selected histone marks on the promoters of human SOX1 and SOX2 genes. We demonstrate differences in histone signatures of SOX1, SOX2 and SOX3 genes. Considering the importance of SOXB1 genes in the process of neural differentiation, the present study contributes to a better understanding of epigenetic mechanisms implicated in the regulation of pluripotency maintenance and commitment towards the neural lineage.

Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells

MicroRNAs (miRNAs) regulate critical cell processes, such as apoptosis, proliferation, and development. However, the role of miRNAs in embryonic stem cell (ESC) neural differentiation induced by retinoic acid (RA) and factors that govern neural directional differentiation remain poorly understood. In this study, we demonstrated that miR-219 is sufficient in promoting mouse ESCs to undergo neural differentiation. We discovered that Foxj3 and Zbtb18, two target genes of miR-219, are not able to determine the process of RA-induced differentiation, however they prevent ESCs from differentiating into neural cells. We identified four downstream genes, namely, Olig1, Zic5, Erbb2, and Olig2, which are essential to the gene interaction networks for neural differentiation. These data explain the mechanism of RA-induced neural differentiation of mESCs on the basis of miRNAs and support the crucial role of miR-219 in neurodevelopment.

Impaired neural differentiation potency by retinoic acid receptor-α pathway defect in induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are reprogrammed from somatic cells via ectopic gene expression and, similarly to embryonic stem cells (ESCs), possess powerful abilities to self-renew and differentiate into cells of various lineages. However, the neural differentiation potency of iPSCs remains unknown. In this study, we demonstrated the neural differentiation ability of iPSCs compared with ESCs using an retinoic acid (RA) induction system. The neural differentiation efficiency of iPSCs was obviously lower than that of ESCs. Retinoic acid receptor-α (RARα) was critical in the RA-induced neural differentiation of iPSCs, and the effect of RARα was confirmed by applying a specific RARα antagonist ER50891 to ESCs. These findings indicate that iPSCs do not possess the complete properties that ESCs have.

Prdm12 Is Induced by Retinoic Acid and Exhibits Anti-proliferative Properties through the Cell Cycle Modulation of P19 Embryonic Carcinoma Cells

The Prdm (PRDI-BF1-RIZ1 homologous) family is involved in cell differentiation, and several Prdms have been reported to methylate histone H3 by intrinsic or extrinsic pathways. Here, we report that Prdm12 recruits G9a to methylate histone H3 on lysine 9 through its zinc finger domains. Because of the expression of Prdm12 in the developmental nervous system, we investigated the role of Prdm12 on P19 embryonic carcinoma cells as a model system for neurogenesis. In P19 cells, Prdm12 is induced by Retinoic acid (RA). Overproduction of Prdm12 in P19 cells impairs cell proliferation and increases the G1 population accompanied by the upregulation of p27. In contrast, the knockdown of Prdm12 increases the number of cells in a suspension culture of RA-induced neural differentiation. Both the PR domain and zinc finger domains are required for the anti-proliferative activity of Prdm12. While the data in this study is based on in vitro models, the results suggest that Prdm12 is induced by the RA signaling in vivo, and may regulate neural differentiation during animal development.

Retinoic acid dependent histone 3 demethylation of the clusteredHOXgenes during neural differentiation of human embryonic stem cells

Gene activation of HOX clusters is an early event in embryonic development. These genes are highly expressed and active in the vertebrate nervous system. Based on the presence of retinoic acid response elements (RAREs) in the regulatory region of many of the HOX genes, it is deduced that retinoic acid (RA) can influence epigenetic regulation and consequently the expression pattern of HOX during RA-induced differentiation of embryonic model systems. In this investigation, the expression level as well as the epigenetic regulation of several HOX genes of the 4 A-D clusters was analyzed in human embryonic stem cells, and also through their neural induction, in the presence and absence of RA. Expression analysis data significantly showed increased mRNA levels of all examined HOX genes in the presence of RA. Epigenetic analysis of the HOX gene regulatory regions also showed a significant decrease in methylation of histone H3K27 parallel to an absolute preferential incorporation of the demethylase UTX rather than JMJD3 in RA-induced neural differentiated cells. This finding clearly showed the functional role of UTX in epigenetic alteration of HOX clusters during RA-induced neural differentiation; the activity could not be detectable for the demethylase JMJD3 during this developmental process.

Involvement of NOS3 in RA‐Induced neural differentiation of human NT2/D1 cells

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5' regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter (-734 to -989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.

Expression analysis of Fgf8a &amp; Fgf8b in early stage of P19 cells during neural differentiation

Fgf8 is a member of the fibroblast growth factor (FGF) family that plays an important role in early neural development. Cellular aggregation and retinoic acid (RA) are needed for mouse embryonic carcinoma (EC) P19 cell neural differentiation. We have examined the Fgf8 gene in P19 cells during neural differentiation and identified 2 alternatively spliced Fgf8 isoforms, Fgf8a and Fgf8b, among the 8 known splicing isoforms in mammals. The expression of Fgf8a and Fgf8b mRNAs transiently and rapidly increased in the early stage of P19 cells during RA-induced neural differentiation, followed by a decline in expression. The relative amount of Fgf8b was clearly higher than that of Fgf8a at different time-points measured within 24h after RA treatment. Increased Fgf8b mRNA expression was cellular-aggregation dependent. The results demonstrated that cellular-aggregation-induced Fgf8b, but not Fgf8a, may play a pivotal role in early neural differentiation of P19 cells.

Requirement of Retinoic Acid Receptor Isotypes α, β, and γ during the Initial Steps of Neural Differentiation of PCC7 Cells

Retinoic acid (RA) is indispensable for morphogenesis and differentiation of several tissues, including the nervous system. The requirement of the RA receptor (RAR) isotypes alpha, beta, and gamma and the putative role of retinoid X receptor-(RXR) signaling in RA-induced neural differentiation, was analyzed. For this compound-selective retinoids and the murine embryonal carcinoma cell line PCC7, a model system for RA-dependent neural differentiation was used. The present paper shows that proliferating PCC7 cells primarily express RXRalpha and RARalpha, lower levels of RXRbeta, and barely detectable amounts of RARbeta, RARgamma, and RXRgamma. At receptor-selective concentrations, only a RARalpha or RARgamma agonist induced the typical tissue-like differentiation pattern consisting of neuronal and nonneuronal cells. Differentiation-associated processes, such as the down-regulation of Oct4, up-regulation of certain nuclear receptors and proneuronal genes, and the induction of neuronal markers could be triggered by receptor-selective concentrations of a RARalpha-, beta-, or gamma-selective agonist, although with distinct efficacy. The differences are only partially explained by the distinct RARalpha, beta, and gamma expression levels and the dissociation constants for the bound retinoids, suggesting differential requirement of RAR isotypes during the initial stages of neural differentiation of PCC7 cells.

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.

Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells.

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

Retinoic acid-induced differentiation into astrocytes and glutamatergic neurons is associated with expression of functional and activable phospholipase D

Phospholipase D (PLD) activity in mammalian cells has been associated with cell proliferation and differentiation. Here, we investigated the expression of PLD during differentiation of pluripotent embryonal carcinoma cells (P19) into astrocytes and neurons. Retinoic acid (RA)-induced differentiation increased PLD1 and PLD2 mRNA levels and PLD activity that was responsive to phorbol myristate acetate. Various agonists of membrane receptors activated PLD in RA-differentiated cells. Glutamate was a potent activator of PLD in neurons but not in astrocytes, whereas noradrenaline and carbachol increased PLD activity only in astrocytes. P19 neurons but not astrocytes released glutamate in response to a depolarizing stimulus, confirming the glutamatergic phenotype of these neurons. These results indicate upregulation of PLD gene expression associated with RA-induced neural differentiation.

Functional analysis of microRNAs during the retinoic acid-induced neuronal differentiation of human NT2 cells.

MicroRNAs (miRNAs) are phylogenetically widespread small RNAs in animals and plants. These small RNAs can regulate a gene expression at a translational level and play roles during the development of C. elegans, D. melanogaster and plants. Although more than two hundred miRNAs have been found in mammals, the target mRNAs of miRNAs is unknown. Recently, we identified Hes1, bHLH transcriptional repressor, as a target of miR-23 in NT2 cells. In this study, we further investigate that the expression of Hes1 is regulated by miR-23 during the retinoic acid (RA)-induced neural differentiation of NT2 cells. Reduction in the level of miR-23 by siRNAs resulted in the accumulation of Hes1 in differentiated NT2 cells. Moreover, a reduction in the level of miR-23 by siRNA-miR-23 affected the RA-induced neural differentiation of NT2 cells. Thus, our results indicate that miR-23 has a critical role in the RA-induced neuronal differentiation of NT2 cells.

Activation of c-Jun amino-terminal kinase is required for retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.

A Novel Cis-Acting Element Regulates HES-1 Gene Expression in P19 Embryonal Carcinoma Cells Treated with Retinoic Acid

The regulatory mechanisms of mammalian hairy and Enhancer of split homologue-1 (HES-1) genes were examined in mouse P19 embryonic carcinoma cells (P19 cells). Undifferentiated P19 stem cells expressed a basal level of the HES-1 gene, whereas the expression of this gene was increased upon induction of the cells to a neural cell lineage using retinoic acid (RA). Reporter co-transfection analysis identified an activating region within the upstream promoter region of HES-1 from nucleotides -201 to -172. This activating region, called activating region X (ARX), shows a high GC content and contains both an AP-2 binding motif and a CCAAT box. An electrophoretic mobility shift assay using nuclear proteins extracted from P19 cells showed that ARX forms a specific DNA-protein complex. Importantly, ARX-dependent transcription, as well as ARX-binding activity, was significantly increased in P19 cells treated with RA. These results indicate that ARX transduces signals that up-regulate HES-1 gene expression in response to RA-treatment. Thus, a novel cis-acting element involved in HES-1 gene regulation that plays a role in RA-induced neural differentiation of P19 cells has been identified.