Differentiation Of P19 Cells Research Articles

Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells

Transcription factor Foxa1 plays a critical role during neural differentiation and is induced immediately after retinoic acid (RA)-initiated differentiation of pluripotent P19 embryonal carcinoma cells, correlated with the downregulated expression of pluripotency-related genes such as Nanog. To study whether Foxa1 participates in the repression of pluripotency factors, we expressed Foxa1 ectopically in P19 cells and identified that Nanog was repressed directly by Foxa1. We confirmed that Foxa1 was able to interact with Grg3, which is a transcriptional corepressor that expresses in P19 cells as well as during RA-induced P19 cell differentiation. Knockdown of Foxa1 or Grg3 delayed the downregulation of Nanog expression during RA-induced P19 cell differentiation. Furthermore, we found that Foxa1 recruited Grg3 to the Nanog promoter −2kb upstream region and switched the promoter to an inactive chromatin status represented by typical modifications in histone H3. Together, our results suggested a critical involvement of Foxa1 in the negative regulation of Nanog expression during the differentiation of pluripotent stem cells.

Lin28a regulates neuronal differentiation and controls miR-9 production

microRNAs shape the identity and function of cells by regulating gene expression. It is known that brain-specific miR-9 is controlled transcriptionally; however, it is unknown whether post-transcriptional processes contribute to establishing its levels. Here, we show that miR-9 is regulated transcriptionally and post-transcriptionally during neuronal differentiation of the embryonic carcinoma cell line P19. We demonstrate that miR-9 is more efficiently processed in differentiated than undifferentiated cells. We reveal that Lin28a affects miR-9 by inducing the degradation of its precursor through a uridylation-independent mechanism. Furthermore, we show that constitutively expressed untagged but not GFP-tagged Lin28a decreases differentiation capacity of P19 cells, which coincides with reduced miR-9 levels. Finally, using an inducible system we demonstrate that Lin28a can also reduce miR-9 levels in differentiated P19 cells. Together, our results shed light on the role of Lin28a in neuronal differentiation and increase our understanding of the mechanisms regulating the level of brain-specific microRNAs.

A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis

A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA MB. We developed a color-tunable miRNA-9 MB (ColoR9 MB) to sense miR-9 expression-dependent color change. The ColoR9 MB was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 (BHQ1) at one end and a reference probe with Cy5.5 at the other end. The ColoR9 MB visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from the ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant. These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change.

Dynamic measurements of [IP3]i in cardiac differentiated P19 (CD‐P19) cells with a novel quantum dots (QD) and gold nanoparticles (AuNP) nanobiosensor, QD‐IP3‐AuNP. (1097.4)

We reported the first measurements of the [IP3]i in intact CD‐P19 cells with a novel nanobiosensor for IP3, QD‐IP3‐AuNP. QD‐IP3‐AuNP was fabricated with QD580 (donor) and AuNP (acceptor); an IP3 receptor peptide (RP) was conjugated between them. QD fluorescence increases proportionally to [IP3] bound to the RP. Fluorescence microscope measurements (Ex=488; Em=535 and 580) were made in CD‐P19 cells loaded with (i) QD‐IP3‐AuNP, (ii) Fluo‐3, and (iii) both QD‐IP3‐AuNP and Fluo‐3. Differentiation of P19 cells was induced with 1% DMSO. The responses of [IP3]i and [Ca2+]i were interrogated with two agonists, McN343 (M1) and UTP (P2Y2); and three antagonists, pirenzipine(M1), suramine (P2), and thapsigargin (Ca‐ATPase). In each sample (n=5), cumulative doses (µM) of 0, 0.1, 1, 10, of either agonist were added, following with the applications of 10 µM of one of the antagonists and the repeated cumulative doses of the respective agonist, all at 6 min apart. The dose dependent increases of [IP3]i and [Ca2+]i by both agonists were inhibited by their respective receptor antagonists. For both agonists, thapsigargin had no effect on the induced increases of [IP3]i but further the induced increases in [Ca2+]i. These data suggest that [IP3]i measurements with QD‐IP3‐AuNP in CD‐P19 cells could be a robust platform for cardiac drug discovery and toxicology.

Dynamic measurements of [K+]i in cardiac differentiated P19 cells with a quantum dots (QD) potassium nanobiosensor, K‐QD (1097.5)

We reported the first dynamic measurements of [K+]i in intact differentiated P19 cells using a K‐QD nanobiosensor. K‐QD was synthesized by conjugating a 15‐crown‐5 K+ receptor to water soluble QD emitted at 630 nm. In Vitro spectral characterization of K‐QD confirms the sensitivity and specificity of K‐QD that is operated within the physiological K+ ranged from 4 to 130 mM. Differentiation of P19 cells was induced with 1% DMSO. A photon counting fluorescence microscope system with 405 nm excitation and 630nm emission was used to perform experiments with K‐QD loaded in the differentiated P19 cells using cation liposome. In four studies, 10‐8M to 10‐5M cumulative dose responses of [K+]i of each of the following 10 agents were determined. They were (i) quinidine, TEA and glibenclamide for the inhibition of the efflux of K+; (ii) bumetanide, ouabain, and acetylstrophanthidin for the inhibition of the influx of K+; (iii) levocromokalim and NS1619 for the activation of the K+ efflux; and (iv) nifedipine and verapamil for the inhibition of the calcium channels. Five experiments were performed per agent. The predictable and consistent responses induced by all these agents suggest that the application of K‐QD to determine the [K+]i responses in the cardiac differentiated P19 cells could be a robust platform for K+ channel compound screening, drug discovery and toxicology.Grant Funding Source: NIDDK R44‐DK084600

Neural Epidermal Growth Factor-Like Like Protein 2 (NELL2) Promotes Aggregation of Embryonic Carcinoma P19 Cells by Inducing N-Cadherin Expression

NELL2 was first identified as a mammalian homolog of chick NEL (Neural EGF-like) protein. It is almost exclusively expressed in neurons of the rat brain and has been suggested to play a role in neural differentiation. However, there is still no clear evidence for the detailed function of NELL2 in the differentiation of neurons. In this study, we identified NELL2 function during neural differentiation of mouse embryonic carcinoma P19 cells. Endogenous expression of NELL2 in the P19 cells increased in parallel with the neuronal differentiation induced by retinoic acid (RA). We found that the mouse NELL2 promoter contains RA response elements (RAREs) and that treatment with RA increased NELL2 promoter activity. Transfection of P19 cells with NELL2 expression vectors induced a dramatic increase in cell aggregation, resulting in the facilitation of neural differentiation. Moreover, NELL2 significantly increased N-cadherin expression in the P19 cell. These data suggest that NELL2 plays an important role in the regulation of neuronal differentiation via control of N-cadherin expression and cell aggregation.

Differential Expression Profile of Long Non-coding RNAs during Differentiation of Cardiomyocytes

Many long non-coding RNAs (lncRNAs) are species specific and seem to be less conserved than protein-coding genes. Some of them are involved in the development of the lateral mesoderm in the heart and in the differentiation of cardiomyocytes. The purpose of the study was to investigate the expression profiles of lncRNAs during the differentiation of P19 cells into cardiomyocytes, with a view to studying the biological function of lncRNAs and their involvement in the mechanism of heart development. First, we observed the morphology of P19 cells during differentiation using an inverted microscope. Then, cardiac troponin T (cTnT) expression was detected to validate that the cells had successfully differentiated into cardiac myocytes by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) and western blotting. Lastly, the expression profile of lncRNA genes was obtained using an lncRNA microarray and real-time RT-PCR analyses. The microarray results showed that 40 lncRNAs were differentially expressed, of which 28 were upregulated and 12 were downregulated in differentiated cardiomyocytes. The differentially expressed lncRNAs were further validated. Our results illustrated a critical role of lncRNAs during the differentiation of P19 cells into cardiac myocytes, which will provide the foundation for further study of the biological functions of lncRNAs and the mechanism of heart development.

Analysis of the SWI/SNF chromatin-remodeling complex during early heart development and BAF250a repression cardiac gene transcription during P19 cell differentiation

The regulatory networks of differentiation programs and the molecular mechanisms of lineage-specific gene regulation in mammalian embryos remain only partially defined. We document differential expression and temporal switching of BRG1-associated factor (BAF) subunits, core pluripotency factors and cardiac-specific genes during post-implantation development and subsequent early organogenesis. Using affinity purification of BRG1 ATPase coupled to mass spectrometry, we characterized the cardiac-enriched remodeling complexes present in E8.5 mouse embryos. The relative abundance and combinatorial assembly of the BAF subunits provides functional specificity to Switch/Sucrose NonFermentable (SWI/SNF) complexes resulting in a unique gene expression profile in the developing heart. Remarkably, the specific depletion of the BAF250a subunit demonstrated differential effects on cardiac-specific gene expression and resulted in arrhythmic contracting cardiomyocytes in vitro. Indeed, the BAF250a physically interacts and functionally cooperates with Nucleosome Remodeling and Histone Deacetylase (NURD) complex subunits to repressively regulate chromatin structure of the cardiac genes by switching open and poised chromatin marks associated with active and repressed gene expression. Finally, BAF250a expression modulates BRG1 occupancy at the loci of cardiac genes regulatory regions in P19 cell differentiation. These findings reveal specialized and novel cardiac-enriched SWI/SNF chromatin-remodeling complexes, which are required for heart formation and critical for cardiac gene expression regulation at the early stages of heart development.

MicroRNA Deregulation in Right Ventricular Outflow Tract Myocardium in Nonsyndromic Tetralogy of Fallot

BackgroundTetralogy of Fallot (TOF) is 1 of the most common heart defects in children, and the underlying mechanisms remain largely elusive. MicroRNAs (miRNAs) are a class of regulators of gene expression and are increasingly recognized for their roles in heart development. MethodsTo identify miRNAs abnormally expressed in TOF, microarrays were used to analyze the miRNA expression profiles of 5 samples of myectomy tissues from right ventricular outflow tract (RVOT) obstruction of infants with nonsyndromic TOF and 3 age-matched normal RVOT tissues. ResultsIn total, 41 candidate miRNAs were identified. To further validate the microarray results, the 41 miRNAs were detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in a larger independent population of tissue samples, including 21 from patients with TOF and 6 from normal controls; it was found that 18 miRNAs were expressed at significantly different levels. Bioinformatic analysis revealed that these miRNAs targeted a network of genes involved in heart development and human congenital heart diseases. Further in vitro studies indicated that upregulation of miR-424/424* promoted proliferation and inhibited migration of primary embryonic mouse cardiomyocytes, whereas miR-222 promoted cardiomyocyte proliferation and reduced the cardiomyogenic differentiation of P19 cells. The 3'UTR (3' untranslated region) luciferase assay revealed that miR-424/424* suppressed the expression of HAS2 and NF1, and their mRNAs were underexpressed in the RVOT myocardial tissues of TOF. ConclusionsEighteen miRNAs were identified as being deregulated in RVOT myocardial tissues from infants with nonsyndromic TOF, and in vitro experiments indicated that miR-424/424* and miR-222 are involved in cardiomyocyte proliferation and migration and the cardiomyogenic differentiation of P19 cells.

MicroRNA133 and microRNA499 exert synergistic effect on cardiac differentiation

Purpose: It has been demonstrated that miR1 and miR133 are involved in cardiac development and stem cell differentiation, while miR499 enhances the differentiation of embryonic and adult stem cells into cardiomyocytes (CMC). However, it is currently unknown if these miRNA may act synergistically to improve differentiation efficiency. Methods: Mouse P19 cells transduced with a viral vector expressing GFP under the control of a cTnI promoter were primed with 0.5% of DMSO and used as cardiogenic differentiation model (P19 CTRL). MiR499 was transiently over-expressed in P19 cells individually or together with miR1 or miR133. Moreover, we differentiated P19 cells with pre-miR precursors without DMSO. Cardiac differentiation was quantified by: 1) count of spontaneous beating areas; 2) qPCR of the cardiac markers Nkx2.5, GATA4, MEF2c, MLC2v, Cx43 and cTnT; 3) Western Blot and ICC of cTnT and Cx43; 4) measure of inducibility of RyR-mediated Ca++ transients in P19 exposed to caffeine; 5) recording of mechanical activity after Ca++ depletion and in the presence of nifedipine or ryanodine. Results: The overexpression of miR499 alone increased the number of beating CMC by 1.7 fold compared with P19 CTRL (p<0.05). The association of miR499+133, but not miR499+1, exerted synergistic effects further increasing the number of beating CMC (2.02 fold vs. P19 CTRL, p<0.01). qPCR showed that miR499+133 enhance the expression of GATA4, Nkx2.5, Cx43 and cTnT compared with P19 CTRL (p<0.01). WB and ICC for Cx43 and cTnT confirmed these findings. Caffeine responsiveness was increased 2.6 fold by miR499+133 compared with P19 CTRL (32.5 vs 12.5%, p<0.05). Cyclic contractions were reversibly abolished by extracellular Ca++ depletion and by both nifedipine and ryanodine. Strikingly, the percentage of responsive cells to caffeine was significantly higher in P19 cells treated with pre-miR499+133 but no DMSO compared with P19 CTRL (p<0.05). PCR and WB demonstrated that the over-expression of early and late cardiac genes in P19 cells treated with miRNA precursors and not exposed to DMSO and ICC showed the expression of Cx43 and cTnT proteins, SERCA2a and Cav1.2. Conclusions: Our results show that miR133 and miR499 play synergistic effect in cardiac differentiation to the point that no chemical trigger is required. Our findings may contribute to improve the efficiency of stem cell therapy for cardiac repair.

RING finger protein 10 regulates retinoic acid‐induced neuronal differentiation and the cell cycle exit of P19 embryonic carcinoma cells

Rnf10 is a member of the RING finger protein family. Recently, a number of RING finger proteins were reported to be involved in neuronal differentiation, development, and proliferation. In this study, we observed that the mRNA levels and protein expression of Rnf10 increase significantly upon the retinoic acid-induced neuronal differentiation of P19 cells. Knockdown of Rnf10 by RNA interference significantly impaired neuronal differentiation of P19 cells by attenuating the expression of neuronal markers. Cell cycle profiling revealed that Rnf10-depleted cells were unable to establish cell cycle arrest after RA treatment. In agreement with flow cytometry analysis, increased cell proliferation was observed after RA induction in Rnf10 knockdown cells as determined by a BrdU incorporation assay. Moreover, like Rnf10, the mRNA levels and protein expression of p21 and p27 also increased upon RA induction. Rnf10 knockdown only resulted in a reduction of p21 expression, while p27 and p57 expression remained unchanged, indicating that Rnf10 may regulate cell cycle exit through the p21 pathway. Ectopic p21 expression partially rescued the effect of Rnf10 depletion on the neuronal differentiation of P19 cells. Collectively, these results showed that increase in Rnf10 expression upon RA induction is necessary for the positive regulation of cyclin kinase inhibitor p21 expression, which leads to cell cycle arrest and is critical for neuronal differentiation.

Histone H2B Ubquitination Regulates Retinoic Acid Signaling through the Cooperation of ASXL1 and BAP1

Despite the importance of retinoic acid (RA) signaling and histone monoubiquitination in determining cell fate, the underlying mechanism linking the two processes is poorly explored. We describe that additional sex comb-like 1 (ASXL1) represses RA receptor activity by cooperating with BRCA1-associated protein 1 (BAP1), which contains the ubiquitin C-terminal hydrolase (UCH) domain. Both the UCH- and ASXL1-binding domains of BAP1 were required for cooperation. In contrast to Drosophila BAP1, mammalian BAP1 cleaved ubiquitin from histone H2B. As supported by BAP1 mutants, ASXL1 was critical for BAP1 recruitment to chromatin and its activation therein. ASXL1 requirement was supported using Asxl1 null mice embryonic fibroblasts. Both ASXL1 and BAP1 were downregulated during RA-induced P19 cell differentiation with concomitant increase of ubiquitinated H2B, leading to activation of Hox genes. Our data demonstrate the critical role of ASXL1 cooperation with BAP1 in cell differentiation through the regulation of RA signaling associated with H2B ubiquitination.

The Elongation Complex Components BRD4 and MLLT3/AF9 Are Transcriptional Coactivators of Nuclear Retinoid Receptors

Nuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation.

Myosin VI Reduces Proliferation, but Not Differentiation, in Pluripotent P19 Cells

BackgroundWe have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2′-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.Methodology/Principal FindingsStable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.Conclusions/SignificanceThese results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells.

Csn3 Gene Is Regulated by All-Trans Retinoic Acid during Neural Differentiation in Mouse P19 Cells

κ-Casein (CSN3) is known to play an essential role in controlling the stability of the milk micelles. We found that the expression of Csn3 was induced by all-trans retinoic acid (ATRA) during neural differentiation in P19 embryonal carcinoma cells from our study using DNA microarray. In this paper, we describe the detailed time course of Csn3 expression and the induction mechanism of Csn3 transcription activation in this process. The Csn3 expression was induced rapidly and transiently within 24 h of ATRA treatment. Retinoic acid receptor (RAR)-specific agonists were used in expression analysis to identify the RAR subtype involved upregulation of Csn3; a RARα-specific agonist mimicked the effects of ATRA on induction of Csn3 expression. Therefore, RARα may be the RAR subtype mediating the effects of ATRA on the induction of Csn3 gene transcription in this differentiation-promoting process of P19 cells. We found that the promoter region of Csn3 contained a typical consensus retinoic acid response element (RARE), and this RARE was necessary for ATRA-dependent transcriptional regulation. We confirmed that RARα bound to this RARE sequence in P19 cells. These findings indicated that the Csn3 expression is upregulated via ATRA-bound RARα and binding of this receptor to the RARE in the Csn3 promoter region. This will certainly serve as a first step forward unraveling the mysteries of induction of Csn3 in the process of neural differentiation.

Molecular mechanism of cardiac differentiation in P19 embryonal carcinoma cells regulated by Foxa2

To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism. P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined. P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells. Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.

Radioisotope imaging of microRNA-9-regulating neurogenesis using sodium iodide sympoter

Since microRNAs (miRNA, miR) are known to be critical in various cellular processes and diseases, non-invasive molecular imaging system for miRNA is very important for imaging cellular therapy and disease diagnosis. In this study, we developed a radionuclide imaging system for miR-9 using sodium iodide symporter (NIS). During neuronal differentiation of P 19 cells induced by the treatment of retinoic acid (RA), in vitro and in vivo imaging demonstrated that the expression and activity of NIS from the miR-9 NIS reporter gene was clearly repressed by the increased expression and functional activity of miR-9 that bound with the target sequences in the NIS reporter gene and resulted in destabilized the transcriptional activity of NIS gene, compared with the undifferentiated P19 cells. The decreased activity of NIS from the differentiated P19 cells resulted in low uptake of radionuclide and decreased radioisotope signals. The NIS reporter gene-based miRNA imaging system showed a great specificity of imaging miRNA biogenesis during cellular developments. The miRNA NIS reporter gene will provide high sensitive imaging for visualizing miRNA-regulating cellular developments and diseases.

Effects of miR-19b Overexpression on Proliferation, Differentiation, Apoptosis and Wnt/β-Catenin Signaling Pathway in P19 Cell Model of Cardiac Differentiation In Vitro

MicroRNA (miR)-19b is part of the miR-17-92 cluster associated with cardiac development. Here, we investigated the effects of overexpressing miR-19b on proliferation, differentiation, apoptosis, and regulation of the Wnt/β-catenin signaling pathway in the multipotent murine P19 cell line that can be induced to undergo cardiogenesis. P19 cells were transfected with the miR-19b plasmid or empty vector, and miR-19b overexpression was verified by Quantitative Real-Time PCR (qPCR). The miR-19b or vector control stable cell lines were selected using Blasticidin S HCl, and their proliferation, cell cycle, and apoptosis levels were analyzed using the Cell Counting Kit-8 and flow cytometry. P19 cell differentiation markers, apoptosis-related genes (bax, bcl-2), and Wnt/β-catenin signaling pathway-related genes were detected by qPCR, the corresponding proteins by Western blot. Expression of the Wnt pathway and differentiation marker proteins was also verified by immunofluorescence. Morphological changes associated with apoptosis were observed by electron microscopy and Hoechst staining. On the basis of these results, we demonstrated that miR-19b overexpression promoted proliferation and differentiation but inhibited apoptosis in P19 cells; Wnt and β-catenin expressions were decreased, while that of GSK3β was increased with miR-19b overexpression. Overexpression of miR-19b inhibited activation of the Wnt/β-catenin signaling pathway in P19 cells, which may regulate cardiomyocyte differentiation. Our findings may bring new insights into the mechanisms underlying cardiac diseases and suggest that miR-19b is a potential new therapeutic target for cardiovascular diseases.

Epigenetic Regulation of Dpp6 Expression by Dnmt3b and Its Novel Role in the Inhibition of RA Induced Neuronal Differentiation of P19 Cells

DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.

Role of COUP‐TFI during retinoic acid‐induced differentiation of P19 cells to endodermal cells

Retinoic acid (RA) is a positive regulator of P19 cell differentiation. Silencing of pre-B cell leukemia transcription factors (PBXs) expression in P19 cells (AS cells) results in a failure of these cells to differentiate to endodermal cells upon RA treatment. Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) is an orphan member of the steroid-thyroid hormone superfamily. RA treatment of wild type P19 cells results in a dramatic increase in the expression of COUP-TFI; however, COUP-TFI mRNA levels fail to be elevated upon RA treatment of AS cells indicating that PBX expression is required for elevation in COUP-TFI expression. To study the role of COUP-TFI during RA-dependent differentiation of P19 cells, AS cells that inducibly express various levels of COUP-TFI were prepared. Exogenous expression of COUP-TFI in AS cells, in a dose-dependent fashion, leads to growth inhibition, modest cell cycle disruption, and early apoptosis. Furthermore, AS cells can overcome the blockage in RA-dependent differentiation to endodermal cells when either pharmacological levels of COUP-TFI are expressed or a combination of both the expression of physiological levels of COUP-TFI and RA treatment. Additionally, the mRNA level of several pluripotency associated genes including OCT-4, DAX-1, and SF-1 in the COUP-TFI expressing AS cells are reduced. Moreover, analysis of the expression of primary RA response genes indicates that COUP-TFI is involved in the regulatory modulation of the expression of at least two genes, CYP26A1 and HoxA1. These studies demonstrate that COUP-TFI functions as a physiologically relevant regulator during RA-mediated endodermal differentiation of P19 cells.