Differentiation Of P19 Cells Research Articles

Suppression of Sin3A activity promotes differentiation of pluripotent cells into functional neurons

Sin3 is a transcriptional corepressor for REST silencing machinery that represses multiple neuronal genes in non-neuronal cells. However, functions of Sin3 (Sin3A and Sin3B) in suppression of neuronal phenotypes are not well characterized. Herein we show that Sin3A knockdown impedes the repressive activity of REST and enhances differentiation of pluripotent P19 cells into electrophysiologically active neurons without inducing astrogenesis. It is also found that silencing Sin3B induces neurogenesis of P19 cells with a lower efficiency than Sin3A knockdown. The results suggest that Sin3A has a more profound effect on REST repressive machinery for silencing neuronal genes in P19 cells than Sin3B. Furthermore, we show that a peptide inhibitor of Sin3A-REST interactions promotes differentiation of P19 cells into functional neurons. Observations made in studies using genetic deletion and a synthetic inhibitor suggests that Sin3A plays an important role in the repression of neuronal genes by the REST regulatory mechanism.

MicroRNA-1 upregulation promotes myocardiocyte proliferation and suppresses apoptosis during heart development

Previous studies have investigated the role of microRNAs (miRs) in heart development to reveal the miRNA mechanism of action in congenital heart disease (CHD) in children. The present study aimed to investigate the role of miR‑1 in heart development in P19 cells. The mRNA level for miR‑1 in P19 cells was detected before or after cardiomyocyte differentiation, using reverse transcription‑quantitative polymerase chain reaction analysis. Expression of cardiomyocyte differentiation markers was also analyzed. The effect of miR‑1 overexpression on the viability and apoptosis of differentiated P19 cells was assessed using MTT and AnnexinV‑FITC assays, respectively. Furthermore, the effects of miR-1 on expression of markers of cell proliferation and apoptosis were also analyzed in differentiated P19 cells using western blotting. The results demonstrated that P19 cells were successfully differentiated into cardiomyocytes, and that endogenous miR‑1 expression was significantly decreased in differentiated P19 cells compared with undifferentiated P19 cells. Overexpression of miR‑1 resulted in increased viability in differentiated P19 cells and decreased apoptosis, compared with the normal control. In addition, expression of heart and neural crest derivatives expressed transcript2 (Hand2) was increased in differentiated cells with miR‑1 overexpressed compared with normal cells, while caspase‑3 cleavage was decreased by miR‑1 overexpression. In conclusion, the present study suggested that miR-1 upregulation may be important in regulating cell proliferation and apoptosis in P19 differentiated cardiomyocytes by increasing Hand2 expression and suppressing caspase‑3 cleavage. The present study aimed to provide a theoretical basis for the explanation of the mechanism of CHD and investigate miR‑1 as a potential therapeutic target for its clinical treatment.

Extracellular Signal-regulated Kinases (ERKs) Phosphorylate Lin28a Protein to Modulate P19 Cell Proliferation and Differentiation

Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process.

Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1.

Since the discovery of μ-opioid receptor (MOR) gene two decades ago, various regulatory factors have been shown to interact with the MOR promoter and modulate transcript levels. However, the majority of early transcriptional studies on MOR gene have not addressed how intracellular signaling pathways mediate extracellular modulators. In this study, we demonstrate that MOR epigenetic regulation requires multiple coordinated signals converging at the MOR promoter, involving mitogen-activated protein kinase (MAPK) activation and mitogen- and stress-activated protein kinase 1 (MSK1)-ranges of intracellular signaling pathways similar to those activated by opioid agonists. Inhibiting p38 MAPK or extracellular signal-regulated kinase (ERK) 1/2 MAPK (upstream activators of MSK1) reduced MOR expression levels; accordingly, the functional role of MSK1, but not MSK2, was demonstrated using genetic approaches. However, for maximal MSK1 effect, an open chromatin configuration was required, because in vitro CpG methylation of the MOR promoter abolished MSK1 activity. Finally, endogenous MSK1 levels concomitantly increased to regulate MOR gene expression during neuronal differentiation of P19 cells, suggesting a conserved role of this kinase in the epigenic activation of MOR in neurons. Taken together, our findings indicate that the expression of MOR gene requires the activity of intracellular signaling pathways that have been implicated in the behavioral outcomes of opioid drugs, which suggests that an autoregulatory mechanism may function in opioid systems.

The glycosylation stoichiometry of EWS species in neuronal cells.

Although Ewing sarcoma protein (EWS) is known to be glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc), the dynamics and stoichiometry of its glycosylation remain obscure. Here, we report a dynamic change in the glycosylation stoichiometry of EWS species during neuronal differentiation of embryonic carcinoma P19 cells. Our findings suggest that O-GlcNAc glycosylation participates in the regulation of EWS functions in neuronal cells.

Expression and Regulation of Tal2 during Neuronal Differentiation in P19 Cells.

T-cell acute lymphocytic leukemia 2 (Tal2) is a gene encoding a member of the basic helix-loop-helix transcription factor family, which is essential for the normal development of the mouse brain. We found that Tal2 was induced during neural differentiation in P19 cells, which are pluripotent mouse embryonal carcinoma cells that differentiate into the neural lineage upon both exposure to all-trans retinoic acid (atRA) and the formation of cell aggregation. Tal2 expression during neural differentiation in P19 cells was detected within 3 h after induction with atRA and retinoic acid receptor α (RARα). The atRA-RARα complex is known to bind to a characteristic retinoic acid response element (RARE) located in the promoter of target genes. We found a RARE-like element in the intron of Tal2. We also found a TATA-box-like element in the 5' region. The TATA-box-like element functioned as a core promoter, and TATA- box binding protein bound to this element upstream of Tal2 in P19 cells. The RARE-like element responded to atRA signaling that activated the transcription, and RARα was bound to this element in the intron of Tal2 in P19 cells. Furthermore, the interaction between these elements on Tal2 was confirmed in a chromatin immunoprecipitation assay. Because the neural differentiation of P19 cells mimics in part the development of the nervous system, P19 cells are useful for studying the mechanism underlying the role of Tal2 in neural differentiation. Further work is underway to clarify the function of Tal2 in neural differentiation using the differentiation system of P19 cells.

Bioinformatics characteristics of lncRNA-uc.167 and its temporal and spatial expression pattern for mouse embryonic development

Objective To explore the basic biological characteristics of lncRNA-uc.167, and its spatial distribution, temporal expression pattern during the mouse embryonic development. Methods The UCSC genome browser of ENCODE was used to analyze preliminary bioinformatics of lncRNAs.Real-time (RT)-PCR was applied to detect the expression of uc.167 and neighboring genes in the embryonic mouse heart in different stages (P7.5, P11.5, P14.5, P18.5). Dimethyl sulphoxide was used to induce P19 cell differentiation into the cardiomyocytes.RT-PCR was applied to detect the expression changes in uc.167 and neighboring genes on differential day 0, 4, 6, 8 and 10. Results Full-length of human uc.167 was 201 bp, and human uc.167 was located in the genome 5q14.3 (chr5: 88179623-88179824, GRCh37/hg19). uc.167 mainly expressed in the ventricular muscle tissue.The expression of uc.167 was gradually decreased in the mouse embryonic heart development process(P7.5: 1.000±0.100, P11.5: 0.714±0.107, P14.5: 0.393±0.043, P18.5: 0.125±0.013), while the expression of its neighboring Mef2c gene was gradually increased(P7.5: 1.081±0.118, P11.5: 2.340±0.351, P14.5: 3.958±0.542, P18.5: 9.361±0.722), which showed an opposite trend.The expression of uc.167 during P19 cell differentiation into cardiomyocytes showed a an increase at first and then a decrease pattern, and the highest level expression of uc.167 was on differential day 4(d0: 1.071±0.117, d4: 4.714±0.501, d6: 3.572±0.414, d8: 2.550±0.314, d10: 0.786±0.085). The expression of neighboring gene Mef2c was in an opposite trend(d0: 1.012±0.041, d4: 0.353±0.037, d6: 2.470±0.329, d8: 6.706±0.682, d10: 7.765±0.705). Conclusions It is suggested that uc.167 may take part in the process of embryonic heart development and may play a role through negatively regulating its neighboring gene Mef2c. Key words: Bioinformatics analysis; Cardiac development; LncRNA

Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells.

Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), which controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. The surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the observed effect of Corexit-EC9527A in the cell.

A potent and selective small molecule inhibitor of sirtuin 1 promotes differentiation of pluripotent P19 cells into functional neurons.

Sirtuin 1 (SIRT1) is known to suppress differentiation of pluripotent/multipotent cells and neural progenitor cells into neurons by blocking activation of transcription factors critical for neurogenesis. EX-527 is a highly selective and potent inhibitor against SIRT1 and has been used as a chemical probe that modulates SIRT1-associated biological processes. However, the effect of EX-527 on neuronal differentiation in pluripotent cells has not been well elucidated. Here, we report an examination of EX-527 effects on neurogenesis of pluripotent P19 cells. The results showed that EX-527 greatly accelerated differentiation of P19 cells into neurons without generation of cardiac cells and astrocytes. Importantly, neurons derived from P19 cells treated with EX-527 generated voltage-dependent sodium currents and depolarization-induced action potentials. The findings indicate that the differentiated cells have electrophysiological properties. The present study suggests that the selective SIRT1 inhibitor could have the potential of being employed as a chemical inducer to generate functionally active neurons.

MiR-20a regulates proliferation, differentiation and apoptosis in P19 cell model of cardiac differentiation by targeting Smoothened

ABSTRACTMicroRNA (miR)-20a, a member of the miR-17-92 cluster related to cardiac development, was obviously downregulated in myocardially differentiated P19 cells compared with normal P19 cells. Smoothened (SMO) is a member of the Hh pathway. Hh signaling induces cardiac differentiation in P19 cells, and SMO mediates the Hh pathway during embryonic development. Using bioinformatic prediction software Targetscan (http://www.targetscan.org/), PicTar (http://pictar.bio.nyu.edu), and miRBase (http://microrna.sanger.ac.uk/), miR-20a and the 3′-untranslated region (3′-UTR) of SMO mRNA were predicted to have complementary binding regions. Accordingly, we inferred that miR-20a might act as a regulator of SMO, and regulate proliferation, differentiation and apoptosis in P19 cells. We determined the expression of miR-20a, SMO and marker proteins of cardiomyocytes (cTnT, GATA4 and desmin) by quantitative real-time PCR (qRT-PCR) and western blot assays, and found that P19 cells had differentiated into cardiomyocytes successfully at differentiation day 10, and downregulation of miR-20a and upregulation of SMO existed in myocardially differentiated P19 cells. Cell proliferation, differentiation and apoptosis detection showed that miR-20a upregulation inhibited proliferation and differentiation and enhanced apoptosis in P19 cells. Moreover, we verified that miR-20a directly targeted SMO and knockdown of SMO and miR-20a overexpression had similar effects on P19 cell proliferation, differentiation and apoptosis, which verified the speculation that miR-20a inhibits proliferation and differentiation and enhances apoptosis in P19 cells by directly targeting SMO. Our results suggest that miR-20a may be a potential target against congenital heart diseases.

MiR-30c regulates proliferation, apoptosis and differentiation via the Shh signaling pathway in P19 cells.

MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that suppress protein expression by binding to the 3′ untranslated regions of their target genes. Many studies have shown that miRNAs have important roles in congenital heart diseases (CHDs) by regulating gene expression and signaling pathways. We previously found that miR-30c was highly expressed in the heart tissues of aborted embryos with ventricular septal defects. Therefore, this study aimed to explore the effects of miR-30c in CHDs. miR-30c was overexpressed or knocked down in P19 cells, a myocardial cell model that is widely used to study cardiogenesis. We found that miR-30c overexpression not only increased cell proliferation by promoting cell entry into S phase but also suppressed cell apoptosis. In addition, we found that miR-30c inhibited dimethyl sulfoxide-induced differentiation of P19 cells. miR-30c knockdown, in contrast, inhibited cell proliferation and increased apoptosis and differentiation. The Sonic hedgehog (Shh) signaling pathway is essential for normal embryonic development. Western blotting and luciferase assays revealed that Gli2, a transcriptional factor that has essential roles in the Shh signaling pathway, was a potential target gene of miR-30c. Ptch1, another important player in the Shh signaling pathway and a transcriptional target of Gli2, was downregulated by miR-30c overexpression and upregulated by miR-30c knockdown. Collectively, our study revealed that miR-30c suppressed P19 cell differentiation by inhibiting the Shh signaling pathway and altered the balance between cell proliferation and apoptosis, which may result in embryonic cardiac malfunctions.

LncRNA-uc.167 influences cell proliferation, apoptosis and differentiation of P19 cells by regulating Mef2c

In our previous study we screened thousands of lncRNAs for their relationship with ventricular septal defect. Among these lncRNAs, uc.167 attracted our attention for its high level of conservation and that it was antisense to the Mef2c gene, which encodes myocyte enhancer factor 2C. This study aims to investigate the role of uc.167 during cardiomyocyte maturation in P19 cells induction and possible mechanism. The uc.167 expression level in human heart tissue of ventricular septum defect (VSD) was evaluated by qRT-PCR. The UCSC database was searched to investigate the bioinformatics of uc.167. We constructed overexpression vector of uc.167 and Mef2c. To detect proliferation and apoptosis, we combined cell cycle analysis and CCK8, Hoechst staining, flow cytometry and caspase-3 assays, respectively. The cardiomyogenesis related RNAs (cTnT, GATA4, and Mef2c) and proteins were detected by qRT-PCR and Western blotting. In this study, we found that uc.167 expression was significantly increased in VSD heart tissues. uc.167 is on the opposite strand to the coding gene Mef2c. The expression model of Mef2c and uc.167 showed an opposite correlation in the embryonic development and process of differentiation of P19 cells into cardiomyocytes. Overexpression of uc.167 inhibited proliferation but promoted apoptosis in P19 cells compared with the vector group, and those relative mRNAs and proteins decreased during the differentiation process. Whereas, co-expression of Mef2c and uc.167 can partially reverse the negative effects of uc.167 on proliferation, apoptosis and differentiation. Taken together, our findings suggest that uc.167 contributes to the development potential of VSD and may constitute a potential therapeutic target in this disease. uc.167 influences cell proliferation, apoptosis and differentiation of P19 cell by regulating Mef2c.

Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin.

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

Inhibition of miR-29c promotes proliferation, and inhibits apoptosis and differentiation in P19 embryonic carcinoma cells.

In our previous study, the upregulation of microRNA (miR)-29c was identified in the mother of a fetus with a congenital heart defect. However, the functional and regulatory mechanisms of miR-29c in the development of the heart remain to be elucidated. In the present study, the role and mechanism of miR-29c inhibition in heart development were investigated in an embryonic carcinoma cell model. Inhibition of miR-29c promoted proliferation, and suppressed the apoptosis and differentiation of P19 cells. It was also demonstrated that Wingless-related MMTV integration site 4 (Wnt4) was a target of miR-29c, determined using bioinformatic analysis combined with luciferase assays. The inhibition of miR-29c stimulated the WNT4/β-catenin pathway, promoting proliferation of the P19 cells, but suppressing their differentiation into cardiomyocytes. Furthermore, the inhibition of miR-29c promoted the expression of B cell lymphoma-2 and inhibited cell apoptosis. These results demonstrate the significance of miR-29c in the process of cardiac development and suggest that miR-29c dysregulation may be associated with the occurrence of CHD. Thus, miR-29c may have therapeutic potential in the future.

Methyl viologen induces neural differentiation on murine P19 cells.

Methyl viologen is a highly effective contact herbicide, a neurotoxic compound and an inducer of reactive oxygen species, which generate oxidative stress in cells. Reactive oxygen species has been known to function as an important messenger in cell differentiation. In this study, we investigated the effect of methyl viologen on neural cell differentiation using pluripotent mouse embryonal carcinoma P19 cells, which reportedly differentiate into neural cells upon exposure to retinoic acid. Methyl viologen, an inducer of intracellular reactive oxygen species, induced the differentiation of P19 cells into neural cells in the presence of neurofilament. Reduced glutathione, an eliminator of reactive oxygen species, also induced neural differentiation in P19 cells. These results suggest that P19 cells differentiate into neural cells, conceivably independent of intracellular reactive oxygen species.

Effect of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.

To explore the effects of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the embryonic carcinoma (P19) cell model of cardiac differentiation. Knockdown of LYRM1 using small interfering RNA (siRNA) was confirmed by quantitative real-time PCR. Cell Counting Kit-8(CCK-8) proliferation assays and cell cycle analysis demonstrated that LYRM1 gene silencing significantly inhibited P19 cell proliferation. Flow cytometry and measurement of their caspase-3 activities revealed that knockdown of LYRM1 increased P19 cell apoptosis. Observation of morphological changes using an inverted microscope and expression analysis of specific differentiation marker genes using quantitative real-time PCR and Western blotting revealed that knockdown of LYRM1 significantly inhibited the differentiation of P19 cells into cardiomyocytes. Furthermore, real-time quantitative PCR applied to detect mitochondrial DNA (mtDNA) copy number implied that there was no significant difference in the LYRM1 knockdown group compared with the control group. Cellular ATP production investigated by luciferase-based luminescence assay was dramatically decreased in differentiated cells transfected with LYRM1 RNAi. Fluorescence microscopy and flow cytometery were used to detect the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) showed that the level of ROS was dramatically increased and MMP was obviously decreased in differentiated cells transfected with LYRM1 RNAi. Collectively, knockdown of LYRM1 promoted apoptosis and suppressed proliferation and differentiation in P19 cells. In addition, knockdown of LYRM1 induced mitochondrial impairment in P19 cells during differentiation, which was reflected by decreased ATP synthesis, lower MMP and increased ROS levels.

5-Azacytidine delivered by mesoporous silica nanoparticles regulates the differentiation of P19 cells into cardiomyocytes

Heart disease is one of the deadliest diseases causing mortality due to the limited regenerative capability of highly differentiated cardiomyocytes. Stem cell-based therapy in tissue engineering is one of the most exciting and rapidly growing areas and raises promising prospects for cardiac repair. In this study, we have synthesized FITC-mesoporous silica nanoparticles (FMSNs) based on a sol-gel method (known as Stöber's method) as a drug delivery platform to transport 5-azacytidine in P19 embryonic carcinoma stem cells. The surfactant CTAB is utilized as a liquid crystal template to self-aggregate into micelles, resulting in the synthesis of MSNs. Based on the cell viability assay, treatment with FMSNs + 5-azacytidine resulted in much more significant inhibition of the proliferation than 5-azacytidine alone. To study the mechanism, we have tested the differentiation genes and cardiac marker genes in P19 cells and found that these genes have been up-regulated in P19 embryonic carcinoma stem cells treated with FMSNs + 5-azacytidine + poly(allylamine hydrochloride) (PAH), with the changes of histone modifications on the regulatory region. In conclusion, with FMSNs as drug delivery platforms, 5-azacytidine can be more efficiently delivered into stem cells and can be used to monitor and track the transfection process in situ to clarify their effects on stem cell functions and the differentiation process, which can serve as a promising tool in tissue engineering and other biomedical fields.

Restoration of miR-98 relieves the inhibitory effect of nicotine on the differentiation of P19 cells into cardiomyocytes.

To study whether miR-98 participates in the effects of nicotine on myocardial differentiation. By western blot, MTT and flow cytometry assays, we found that nicotine suppresses P19 cell differentiation into cardiomyocytes and apoptosis, and promotes proliferation, while restoration of miR-98 relieves the inhibitory effect of nicotine on the P19 cell differentiation. By target prediction analysis and luciferase reporter assay, we observed that miR-98 inhibits the protein expression of Wnt1 by directly acting on the 3'-UTR of Wnt1 mRNA. We assumed that the effect of miR-98 on Wnt1 might alter the activity of the Wnt1/β-catenin signaling pathway and be associated with myocardial differentiation. In summary, nicotine restrains differentiation of P19 cells into cardiomyocytes and decreases the level of miR-98. Restoration of miR-98 relieves the inhibitory effect of nicotine on differentiation of P19 cells via targeting the 3'-UTR of Wnt1, which offers novel insights into our understanding of underlying molecular mechanisms of congenital heart defects.

Possible activation by the green tea amino acid theanine of mammalian target of rapamycin signaling in undifferentiated neural progenitor cells in vitro

We have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR) kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2’-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells.

Sox6 suppression induces RA-dependent apoptosis mediated by BMP-4 expression during neuronal differentiation in P19 cells.

Sox6 is a transcription factor that induces neuronal differentiation in P19 cells; its suppression not only inhibits neuronal differentiation but also induces retinoic acid (RA)-dependent apoptosis of P19 cells. In the present study, we found that Sox6 suppression-induced apoptosis was mediated by activation of caspase 9 and 3. Moreover, we noted a weak leakage of cytochrome c into the cytoplasm from the mitochondria, indicating that apoptosis occurs through a mitochondrial pathway in Sox6-suppressed P19 (P19[anti-Sox6]) cells. Sox6 suppression in the presence of RA also induced the expression and secretion of bone morphogenetic protein 4 (BMP-4). Addition of an anti-BMP-4 antibody for neutralization increased cell viability and led to RA-dependent death of P19[anti-Sox6] cells. Our results indicate that Sox6 suppression induces RA-dependent cell death of P19 cells, mediated by BMP-4 expression and secretion. Normally, high Sox6 expression leads to RA-mediated neuronal differentiation in P19 cells; however, Sox6 deficiency induces production and secretion of BMP-4, which mediates selective cell death. Our findings suggest that Sox6 contributes to cell survival by suppressing BMP-4 transcription during neuronal differentiation.