P19 Embryonal Carcinoma Cells Research Articles

P19-derived neuronal cells express H1, H2, and H3 histamine receptors: a biopharmaceutical approach to evaluate antihistamine agents

Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.

Suppressor of Fused Regulation of Hedgehog Signaling is Required for Proper Astrocyte Differentiation.

Hedgehog signaling is essential for vertebrate development; however, less is known about the negative regulators that influence this pathway. Using the mouse P19 embryonal carcinoma cell model, suppressor of fused (SUFU), a negative regulator of the Hedgehog (Hh) pathway, was investigated during retinoic acid (RA)-induced neural differentiation. We found Hh signaling increased activity in the early phase of differentiation, but was reduced during terminal differentiation of neurons and astrocytes. This early increase in pathway activity was required for neural differentiation; however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of Gli, remained relatively unchanged during differentiation, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hh target genes. Interestingly, these SUFU-deficient cells were unable to differentiate toward neural lineages without RA, and when directed toward these lineages, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout RA-induced differentiation and this was accompanied by the loss of Gli3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, likely acting through Gli3, to reduce Hh signaling during late-stage differentiation.

Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

Wnt and Hh are known signalling pathways involved in neural differentiation and recent work has shown the cell cycle regulator, Never in Mitosis Kinase 2 (Nek2) is able to regulate both pathways. Despite its known function in pathway regulation, few studies have explored Nek2 within embryonic development. The P19 embryonal carcinoma cell model was used to investigate Nek2 and neural differentiation through CRISPR knockout and overexpression studies. Loss of Nek2 reduced cell proliferation in the undifferentiated state and during directed differentiation, while overexpression increased cell proliferation. Despite these changes in proliferation rates, Nek2 deficient cells maintained pluripotency markers after neural induction while Nek2 overexpressing cells lost these markers in the undifferentiated state. Nek2 deficient cells lost the ability to differentiate into both neurons and astrocytes, although Nek2 overexpressing cells enhanced neuron differentiation at the expense of astrocytes. Hh and Wnt signalling were explored, however there was no clear connection between Nek2 and these pathways causing the observed changes to differentiation phenotypes. Mass spectrometry was also used during wildtype and Nek2 knockout cell differentiation and we identified reduced electron transport chain components in the knockout population. Immunoblotting confirmed the loss of these components and additional studies showed cells lacking Nek2 were exclusively glycolytic. Interestingly, hypoxia inducible factor 1α was stabilized in these Nek2 knockout cells despite culturing them under normoxic conditions. Since neural differentiation requires a metabolic switch from glycolysis to oxidative phosphorylation, we propose a mechanism where Nek2 prevents HIF1α stabilization, thereby allowing cells to use oxidative phosphorylation to facilitate neuron and astrocyte differentiation.

Sel1l May Contributes to the Determinants of Neuronal Lineage and Neuronal Maturation Regardless of Hrd1 via Atf6-Sel1l Signaling.

The endoplasmic reticulum (ER) is the primary site of intracellular quality control involved in the recognition and degradation of unfolded proteins. A variety of stresses, including hypoxia and glucose starvation, can lead to accumulation of unfolded proteins triggering the ER-associated degradation (ERAD) pathway. Suppressor Enhancer Lin12/Notch1 Like (Sel1l) acts as a "gate keeper" in the quality control of de novo synthesized proteins and complexes with the ubiquitin ligase Hrd1 in the ER membrane. We previously demonstrated that ER stress-induced aberrant neural stem cell (NSC) differentiation and inhibited neurite outgrowth. Inhibition of neurite outgrowth was associated with increased Hrd1 expression; however, the contribution of Sel1l remained unclear. To investigate whether ER stress is induced during normal neuronal differentiation, we semi-quantitatively evaluated mRNA expression levels of unfolded protein response (UPR)-related genes in P19 embryonic carcinoma cells undergoing neuronal differentiation in vitro. Stimulation with all-trans retinoic acid (ATRA) for 4days induced the upregulation of Nestin and several UPR-related genes (Atf6, Xbp1, Chop, Hrd1, and Sel1l), whereas Atf4 and Grp78/Bip were unchanged. Small-interfering RNA (siRNA)-mediated knockdown of Sel1l uncovered that mRNA levels of the neural progenitor marker Math1 (also known as Atoh1) and the neuronal marker Math3 (also known as Atoh3 and NeuroD4) were significantly suppressed at 4days after ATRA stimulation. Consistent with this result, Sel1l silencing significantly reduced protein levels of immature neuronal marker βIII-tubulin (also known as Tuj-1) at 8days after induction of neuronal differentiation, whereas synaptogenic factors, such as cell adhesion molecule 1 (CADM1) and SH3 and multiple ankyrin repeat domain protein 3 (Shank3) were accumulated in Sel1l silenced cells. These results indicate that neuronal differentiation triggers ER stress and suggest that Sel1l may facilitate neuronal lineage through the regulation of Math1 and Math3 expression.

Decoding the dynamic H3K9cr landscapes during neural commitment of P19 embryonal carcinoma cells

Histone lysine crotonylation (Kcr) is a novel hydrophobic histone acylation modification, and we recently report its crucial roles in neural differentiation. However, it is still unclear how histone Kcr involve in early neural commitment. Here, we systematically investigate the H3K9cr landscapes during neuroectodermal differentiation of pluripotent P19 embryonal carcinoma cells (ECCs). We reveal that the genome-wide changes in H3K9cr favor neural fate specification, and identify potential co-factors binding H3K9cr. We also uncover that H3K9cr collaborates with H3K9ac to regulate gene expression changes. Our results provide novel insights into the epigenetic mechanisms underlying neural commitment.

Hypermethylation of Mest promoter causes aberrant Wnt signaling in patients with Alzheimer\u2019s disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to dementia and behavioral changes. Extracellular deposition of amyloid plaques (Aβ) and intracellular deposition of neurofibrillary tangles in neurons are the major pathogenicities of AD. However, drugs targeting these therapeutic targets are not effective. Therefore, novel targets for the treatment of AD urgently need to be identified. Expression of the mesoderm-specific transcript (Mest) is regulated by genomic imprinting, where only the paternal allele is active for transcription. We identified hypermethylation on the Mest promoter, which led to a reduction in Mest mRNA levels and activation of Wnt signaling in brain tissues of AD patients. Mest knockout (KO) using the CRIPSR/Cas9 system in mouse embryonic stem cells and P19 embryonic carcinoma cells leads to neuronal differentiation arrest. Depletion of Mest in primary hippocampal neurons via lentivirus expressing shMest or inducible KO system causes neurodegeneration. Notably, depletion of Mest in primary cortical neurons of rats leads to tau phosphorylation at the S199 and T231 sites. Overall, our data suggest that hypermethylation of the Mest promoter may cause or facilitate the progression of AD.

Effect of glycosaminoglycan structure on all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells

Glycosaminoglycan polysaccharides are components of animal extracellular matrices and regulate cell functions based on their various sulfation and epimerization pattern structures. The present study aimed to find glycosaminoglycan structures to promote neural differentiation. We investigated the effect of exogenous glycosaminoglycans with well-defined structures on the all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells, which is an ideal model culture system for studying neural differentiation. We found that chondroitin sulfate E and heparin, but not any other glycosaminoglycans, upregulated the expressions of neural specific markers but not a grail specific marker. Chondroitin sulfate E was suggested to function during spheroid formation, however, equimolar concentration of its oligosaccharide did not show promotive effect on the neural differentiation. Another finding was that hyaluronan oligosaccharide mixture markedly downregulated the expressions of a myelin specific marker. These findings suggested that the specific sulfation pattern and/or chain length of exogenous added glycosaminoglycan is important to regulate neural differentiation and myelination.

Adipose Tissue as a Useful Material for the Grafting of Tumorigenic Cells and Juvenile Tissues in Mice

Although the aging process expands the adipose tissue habitation in mice and due to its close association with the female reproductive system, it can be easily exposed surgically under anesthesia when reproductive organs (including ovary, oviduct, and part of the uterus) are pulled and exposed onto the dorsal skin. This study aimed to consider the suitability of adipose tissue as a target for manipulation, particularly for the grafting of cells or small-sized tissue sections due to its ease of handling. Subsequently, 1-2 µL trypan blue injections were administered to the tissues using a breath-controlled micropipette under a dissecting microscope for evaluating the adipose tissue’s potential as a suitable grafting material. It was observed that the injected dye remained at the injection site for at least one day after injecting B16 mouse melanoma and P19 embryonal carcinoma cells. It resulted in the generation of solid tumors surrounding the ovary, oviduct, or uterus with 100% efficiency, as reported by an inspection one and a half months after the injections. When the grafting procedure was carried out for one-fourth of the juvenile pancreas (aged 15 days), an enlarged pancreas with normal morphological configuration (including the formation of insulin-synthesizing cells) was generated, which was observed by an inspection one month after the injections, thus successfully validating our approach for adipose tissue manipulation and furthermore, naming this novel technology as “intra-adipose introduction of cells and tissues”.

Electroactive and antioxidant injectable in-situ forming hydrogels with tunable properties by polyethylenimine and polyaniline for nerve tissue engineering

The injectable in-situ forming electroconductive hydrogels with antioxidant activity are promising candidates for nerve tissue engineering. In this study, we synthesized and developed a gelatin-graft-polyaniline/periodate-oxidized alginate hydrogel through the introduction of branched polyethylenimine (PEI) to improve the rheological properties. Moreover, antioxidant property, electroconductivity and the effect of external electrical stimulus on the nerve cell behavior were investigated. The results showed that by increasing the polyaniline content, the antioxidant activity, pore sizes, and swelling ratio of the hydrogel were increased, while the crosslinking density and storage modulus were decreased. The introduction of PEI accelerated the gelation time, decreased swelling ratio and pore size, and increased the storage modulus and crosslinking density. Cell studies showed that all formulations had supported the viability of P19 embryonic carcinoma cells with the neuritis elongation in the presence of the external electrical-stimulus. Gene expression of the neuronal markers, including Nestin, Pax-6, and β-tubulin III, was increased in all hydrogels; In addition, electrical stimulation significantly elevated the expression of these markers in high polyaniline-content hydrogel compared to the polyaniline-free hydrogel. In conclusion, the results suggest that the prepared injectable electroconductive hydrogels can be a promising approach for neural tissue engineering.

MicroRNA-29c affects zebrafish cardiac development via targeting Wnt4.

As a single cardiac malformation, ventricular septal defect (VSD) is the most common form of congenital heart disease. However, the precise molecular mechanisms underlying VSD are not completely understood. Numerous microRNAs (miRs/miRNAs) are associated with ventricular septal defects. miR-29c inhibits the proliferation and promotes the apoptosis and differentiation of P19 embryonal carcinoma cells, possibly via suppressing Wnt4 signaling. However, to the best of our knowledge, no in vivo studies have been published to determine whether overexpression of miR-29c leads to developmental abnormalities. The present study was designed to observe the effect of miRNA-29c on cardiac development and its possible mechanism in vivo. Zebrafish embryos were microinjected with different doses (1, 1.6 and 2 µmol) miR-29c mimics or negative controls, and hatchability, mortality and cardiac malformation were subsequently observed. The results showed that in zebrafish embryos, miR-29c overexpression attenuated heart development in a dose-dependent manner, manifested by heart rate slowdown, pericardial edema and heart looping disorder. Further experiments showed that overexpression of miR-29c was associated with the Wnt4/β-catenin signaling pathway to regulate zebrafish embryonic heart development. In conclusion, the present results demonstrated that miR-29c regulated the lateral development and cardiac circulation of zebrafish embryo by targeting Wnt4.

Effects of lamellar microstructure of retinoic acid loaded-matrixes on physicochemical properties, migration, and neural differentiation of P19 embryonic carcinoma cells

Abstract In this study, retinoic acid loaded-PLGA-gelatin matrixes were prepared with both freeze-casting and freeze-drying techniques. Herein, the effect of unidirectional microstructure with tunable pores on release profile, cellular adhesion, migration, and differentiation was compared. Morphological observation determined that highly interconnected porous structure can be formed, but lamellar pore channels were observed in freeze-casting prepared constructs. The absorption ratio was increased, and the biodegradation rate was decreased as a function of the orientation of microstructure. The in-vitro release study illustrated non-Fickian release mechanism in both methods, so that erosion has predominated over diffusion. Accordingly, PLGA-gelatin scaffolds prepared with freeze-drying technique showed no adequate erosion due to the rigid structure, while freeze-casting one presented more favorable erosion. Microscopic observations of adhered P19 embryonic cells on the scaffolds showed that the freeze-casting matrixes with unidirectional pores provide a more compatible microenvironment for cell attachments and spreading. Besides, it facilitated cell migration and penetration inside the structure and may act as guidance for neuron growth. Improvement in the expression of neural genes in unidirectionally oriented pores proved the decisive role of contact guidance for nerve healing. It seems that the freeze-cast PLGA-gelatin-retinoic acid scaffolds have initial features for nerve tissue regeneration studies.

Establishment of a Simple Method for Inducing Neuronal Differentiation of P19 EC Cells without Embryoid Body Formation and Analysis of the Role of Histone Deacetylase 8 Activity in This Differentiation.

P19 pluripotent embryonic carcinoma (EC) stem cells are derived from pluripotent germ cell tumours and can differentiate into three germ layers. Treatment of these cells in suspension culture with retinoic acid induces their differentiation into neurons and glial cells. Hence, these cells are an excellent in vitro model to study the transition from the upper blastoderm to the neuroectoderm. However, because of the complex nature of the techniques involved, the results are highly dependent on the skills of the experimenter. Herein, we developed a simple method to induce neuronal differentiation of adherent P19 EC cells in TaKaRa NDiff® 227 serum-free medium (originally N2B27 medium). This medium markedly induced neuronal differentiation of P19 EC cells. The addition of retinoic acid to the NDiff® 227 medium further enhanced differentiation. Furthermore, cells differentiated by the conventional method, as well as the new method, showed identical expression of the mature neuronal marker, neuronal nuclei. To determine whether our approach could be applied for neuronal studies, we measured histone deacetylase 8 (HDAC8) activity using an HDAC8 inhibitor and HDAC8-knockout P19 EC cells. Inhibition of HDAC8 activity suppressed neuronal maturation. Additionally, HDAC8-knockout cell lines showed immature differentiation compared to the wild-type cell line. These results indicate that HDAC8 directly regulates the neuronal differentiation of P19 EC cells. Thus, our method involving P19 EC cells can be used as an experimental system to study the nervous system. Moreover, this method is suitable for screening drugs that affect the nervous system and cell differentiation.

Correction to: Bilobalide Induces Neuronal Differentiation of P19 Embryonic Carcinoma Cells via Activating Wnt/β-Catenin Pathway.

The original version of this article unfortunately contained an error in Figures 1A and 4C.

Yokukansan, a Kampo medicine, enhances the level of neuronal lineage markers in differentiated P19 embryonic carcinoma cells

Yokukansan (YKS), a traditional Japanese Kampo medicine, affects neurological and psychiatric disorders. It ameliorates hippocampal neurogenesis in animals. However, its effect on neuronal cell differentiation remains unclear. Therefore, we investigated the effects of YKS on pluripotent P19 embryonic carcinoma cells as neuronal differentiation model cells.Western blotting and immunocytochemistry revealed that 10 μg/mL YKS treatment during embryoid body formation or neuronal differentiation increased the expression of the neuronal stem cell marker, Nestin, by 1.9-fold and 1.7-fold, respectively, and of the mature neuron marker, NeuN, by 1.5-fold and 1.4-fold, respectively.We examined the effect of YKS on intracellular signaling pathways in P19 cells and found significant elevation in phospho-PDK1 and phospho-mTOR expression (1.1-fold and 1.2-fold, respectively). Therefore, we investigated the effect of PDK1 and mTOR inhibitors on the level of neuronal lineage markers. We found that the mTOR inhibitor significantly abolished the YKS effect on the level of neuronal lineage markers.Moreover, to identify the target(s) of YKS, antibody array analysis that simultaneously detects 16 phosphorylated proteins was performed. YKS significantly upregulated 10 phosphorylated proteins including PDK1, Akt, AMPK, PRAS40, mTOR, p70 S6 kinase, GSK-3α, Bad and ERK1/2 under cell proliferation conditions. These results suggest that YKS simultaneously activates multiple signaling pathways.Thus, we concluded that YKS enhances the level of neuronal lineage markers in differentiated P19 cells, however it does not induce neuronal differentiation. Furthermore, mTOR is the predominant mediator of the YKS effect on these cells.

Retinoic acid receptor gamma is targeted by microRNA-124 and inhibits neurite outgrowth

During brain development, neurite outgrowth is required for brain development and is regulated by many factors. All-trans retinoic acid (RA) is an important regulator of cell growth and differentiation. MicroRNA-124 (miR-124), a brain-specific microRNA, has been implicated in stimulating neurite growth. In this study, we found that retinoic acid receptor gamma (RARG) expression was decreased, whereas miR-124 expression was increased during neural differentiation in mouse Neuroblastoma (N2a) Cells, P19 embryonal carcinoma (P19) cells, and mouse brain, as detected by immunoblotting or RT-qPCR. And we proved that miR-124 inhibited RARG expression by binding to the 3′ UTR of RARG with a luciferase reporter assay. Upregulation of miR-124 (using miR-124 overexpressing plasmid and miR-124 mimic) led to a significant decrease in RARG protein in N2a cells and primary neurons. Therefore, we asked whether and how the miR-124/RARG axis regulates neuronal outgrowth, which is poorly understood. Strikingly, RARG knockdown by shRNA stimulated neurite growth in N2a cells and primary neurons, whereas RARG overexpression (without 3′ UTR) inhibited neurite growth in N2a cells, P19 cells, and primary neurons. Furthermore, RARG knockdown could partially eliminate neurite outgrowth defects caused by the inhibitor of miR-124, while RARG overexpression could reverse the neurite outgrowth enhancing effect of the upregulation of miR-124. Collectively, the data reveal that miR-124/RARG axis is critical for neurite outgrowth. RARG emerges as a new target regulated by miR-124 that modulates neurite outgrowth, providing a novel context in which these two molecules function.

Cell quality control mechanisms maintain stemness and differentiation potential of P19 embryonic carcinoma cells

ABSTRACTGiven the relatively long life of stem cells (SCs), efficient mechanisms of quality control to balance cell survival and resistance to external and internal stress are required. Our objective was to test the relevance of cell quality control mechanisms for SCs maintenance, differentiation and resistance to cell death. We compared cell quality control in P19 stem cells (P19SCs) before and after differentiation (P19dCs). Differentiation of P19SCs resulted in alterations in parameters involved in cell survival and protein homeostasis, including the redox system, cardiolipin and lipid profiles, unfolded protein response, ubiquitin-proteasome and lysosomal systems, and signaling pathways controlling cell growth. In addition, P19SCs pluripotency was correlated with stronger antioxidant protection, modulation of apoptosis, and activation of macroautophagy, which all contributed to preserve SCs quality by increasing the threshold for cell death activation. Furthermore, our findings identify critical roles for the PI3K-AKT-MTOR pathway, as well as autophagic flux and apoptosis regulation in the maintenance of P19SCs pluripotency and differentiation potential.Abbreviations: 3-MA: 3-methyladenine; AKT/protein kinase B: thymoma viral proto-oncogene; AKT1: thymoma viral proto-oncogene 1; ATG: AuTophaGy-related; ATF6: activating transcription factor 6; BAX: BCL2-associated X protein; BBC3/PUMA: BCL2 binding component 3; BCL2: B cell leukemia/lymphoma 2; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CL: cardiolipin; CTSB: cathepsin B; CTSD: cathepsin D; DDIT3/CHOP: DNA-damage inducible transcript 3; DNM1L/DRP1: dynamin 1-like; DRAM1: DNA-damage regulated autophagy modulator 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2, subunit alpha; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; ESCs: embryonic stem cells; KRT8/TROMA-1: cytokeratin 8; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NANOG: Nanog homeobox; NAO: 10-N-nonyl acridine orange; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; OPA1: OPA1, mitochondrial dynamin like GTPase; P19dCs: P19 differentiated cells; P19SCs: P19 stem cells; POU5F1/OCT4: POU domain, class 5, transcription factor 1; PtdIns3K: phosphatidylinositol 3-kinase; RA: retinoic acid; ROS: reactive oxygen species; RPS6KB1/p70S6K: ribosomal protein S6 kinase, polypeptide 1; SCs: stem cells; SOD: superoxide dismutase; SHC1-1/p66SHC: src homology 2 domain-containing transforming protein C1, 66 kDa isoform; SOX2: SRY (sex determining region Y)-box 2; SQSTM1/p62: sequestosome 1; SPTAN1/αII-spectrin: spectrin alpha, non-erythrocytic 1; TOMM20: translocase of outer mitochondrial membrane 20; TRP53/p53: transformation related protein 53; TUBB3/betaIII-tubulin: tubulin, beta 3 class III; UPR: unfolded protein response; UPS: ubiquitin-proteasome system

E2f1 regulates the induction of promyelocytic leukemia zinc finger transcription in neuronal differentiation of pluripotent P19 embryonal carcinoma cells

During brain development, the expression of promyelocytic leukemia zinc finger (Plzf) in neural stem cells is precisely controlled to maintain the balance between neural stem cell self-renewal and differentiation. However, the mechanism underlying transcriptional regulation of Plzf in neural stem cell is still unclear. Herein, using P19 embryonal carcinoma cells as a model, we observed that Plzf expression was induced in the P19-derived embryonic bodies, which enrich neural stem-like cell populations, as demonstrated by the expression of neural stem cell markers, Nestin and Sox2. We then characterized the Plzf promoter and identified two E2f1 binding sites (−755/-751 and −53/-49, the transcription start site was designated as +1) are important for the activation of Plzf promoter. Finally, we found that the induction of Plzf in the neural stem-like cells derived from pluripotent P19 cells is decrease by E2f1 knockdown. Taken together, we conclude that E2f1 is an important transcription factor that regulates Plzf transcription and may involve in maintaining the self-renewal ability of neural stem cells.

SGTb regulates a surface localization of a guidance receptor BOC to promote neurite outgrowth

Neuritogenesis is a critical event for neuronal differentiation and neuronal circuitry formation during neuronal development and regeneration. Our previous study revealed a critical role of a guidance receptor BOC in a neuronal differentiation and neurite outgrowth. However, regulatory mechanisms for BOC signaling pathway remain largely unexplored. In the current study, we have identified Small glutamine-rich tetratricopeptide repeat (TPR)-containing b (SGTb) as a BOC interacting protein through yeast two-hybrid screening. Like BOC, SGTb is highly expressed in brain and P19 embryonal carcinoma (EC) cells differentiated into neuronal cells. BOC and SGTb proteins co-precipitate in mouse brain and differentiated P19 EC cells. Furthermore, BOC and SGTb co-localize in neurites and especially are concentrated at the tip of neurites in various neuronal cells. SGTb depletion attenuates neuronal differentiation of P19 cells through reduction of the surface level of BOC. Additionally, SGTb depletion causes BOC localization at neurite tip, coinciding with decreased p-JNK levels critical for actin cytoskeleton remodeling. The overexpression of SGTb or BOC restores JNK activation in BOC or SGTb-depleted cells, respectively. Finally, SGTb elevates the level of surface-resident BOC in BOC-depleted cells, restoring JNK activation. Taken together, our data suggest that SGTb interacts with BOC and regulates its surface level and consequent JNK activation, thereby promoting neuronal differentiation and neurite outgrowth.

Down-regulation of HaIr1 during induced differentiation of embryonal carcinoma P19 cells

Down-regulation of HaIr1 during induced differentiation of embryonal carcinoma P19 cells

Dynamic spatiotemporal patterns of alternative splicing of an F-actin scaffold protein, afadin, during murine development

An F-actin scaffold protein, afadin, comprises two splice variants called l-afadin (a long isoform) and s-afadin (a short isoform). It is known that in adult tissues, l-afadin is ubiquitously expressed while s-afadin is restrictedly expressed in brain. In cultured cortical neurons, l-afadin potentiates axonal branching whereas s-afadin blocks axonal branching by functioning as a naturally occurring dominant-negative isoform that forms a heterodimer with l-afadin. However, the temporal and spatial expression pattern of s-afadin during development or across multiple tissues and organs has not been fully understood. In this study, using Western blotting and RT-qPCR techniques and the fluorescent splicing reporters, we examined the detailed expression patterns of l- and s-afadin isoforms across various tissues and cell types, including rat organs at developmental stages, primary cultured neuronal and non-neuronal cells prepared from the developing rat brain, and in neurons in vitro generated from P19 embryonal carcinoma (EC) cells. Both mRNA and protein of s-afadin were abundantly expressed in various regions of rat neuronal tissues, and their expression dynamically changed during development in vivo. The expression of s-afadin was also detected in primary rat cortical neurons, but not in astrocytes or fibroblasts, and the neuronal expression increased during neuronal maturation in vitro. The dynamic alternative splicing event of afadin during development was successfully visualized with the newly developed fluorescent splicing reporter plasmids at a single cell level. Moreover, s-afadin was undetectable in undifferentiated EC cells, and the expression was induced upon differentiation of the cells into neurons. These data suggest that spatiotemporal and dynamic alternative splicing produces differential expression patterns of afadin isoforms and that alternative splicing of afadin is controlled by the neuron-specific regulator(s) whose activity is triggered and dynamically altered during neuronal differentiation and maturation.