RESUMO
Mitogen-activated protein kinases (MAPKs) are essential for proper cell functioning as they regulate many molecular effectors. Careful regulation of MAPKs is therefore required to avoid MAPK pathway dysfunctions and pathologies. The mammalian genome encodes about 200 phosphatases, many of which dephosphorylate the MAPKs and bring them back to an inactive state. In this review, we focus on the normal and pathological functions of dual-specificity phosphatase 9 (DUSP9)/MAP kinase phosphatases-4 (MKP-4). This cytoplasmic phosphatase, which belongs to the threonine/tyrosine dual-specific phosphatase family and was first described in 1997, is known to dephosphorylate ERK1/2, p38, JNK and ASK1, and thereby to control various MAPK pathway cascades. As a consequence, DUSP9 plays a major role in human pathologies and more specifically in cardiac dysfunction, liver metabolic syndromes, diabetes, obesity and cancer including drug response and cell stemness. Here, we recapitulate the mechanism of action of DUSP9 in the cell, its levels of regulation and its roles in the most frequent human diseases, and discuss its potential as a therapeutic target.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Animais , Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Cardiopatias/enzimologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Doenças Metabólicas/enzimologia , Redes e Vias Metabólicas , Camundongos , Fosfatases da Proteína Quinase Ativada por Mitógeno/química , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Modelos Moleculares , Células-Tronco Embrionárias Murinas/enzimologia , Neoplasias/enzimologia , Conformação Proteica , Caracteres Sexuais , Distribuição TecidualRESUMO
2,2',4,4'-Tetrabromodiphenyl ether (BDE47) poses potential risks to reproduction and development, but the mechanism of its toxicity has not yet been elucidated. To explore the developmental toxicity of BDE47, mouse embryonic stem cells (mESCs), which are ideal models for testing the developmental toxicity of environmental contaminants in vitro, were exposed to BDE47 (0.04 µM, 1 µM, 25 µM, or 100 µM) for 24 h or 48 h in this study. Our results indicated that BDE47 treatment changed the morphology of mESCs, inhibited cell viability and increased apoptosis. In addition, alkaline phosphatase (AP) staining in mESCs was significantly decreased after BDE47 treatment (25 µM and 100 µM), indicating that BDE47 treatment affected the pluripotency of mESCs. Through a cell immunofluorescence assay, we found that the fluorescence intensities of Oct4, Sox2 and Nanog were all significantly lower in the group treated with the highest BDE47 concentration (100 µM) than in the control group, consistent with the qRT-PCR and Western blot results. The levels of miR-145 and miR-34a, which regulate genes related to cell differentiation, were significantly increased in BDE47-treated mESCs, further clarifying the potential mechanism. Overall, our findings demonstrate that BDE47 exposure upregulates the expression of miR-145 and miR-34a and in turn downregulates the expression of Oct4, Sox2 and Nanog, thereby affecting apoptosis and pluripotency and causing toxicity during embryonic development.
Assuntos
Éteres Difenil Halogenados/toxicidade , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Fosfatase Alcalina/análise , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Camundongos , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Embrionárias Murinas/fisiologiaRESUMO
SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.
Assuntos
Diferenciação Celular , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Embrionárias Murinas/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Linhagem da Célula , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/genética , Camundongos , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Sphingolipids are important structural components of cell membranes and prominent signaling molecules controlling cell growth, differentiation, and apoptosis. Sphingolipids are particularly abundant in the brain, and defects in sphingolipid degradation are associated with several human neurodegenerative diseases. However, molecular mechanisms governing sphingolipid metabolism remain unclear. Here, we report that sphingolipid degradation is under transcriptional control of SIRT1, a highly conserved mammalian NAD+-dependent protein deacetylase, in mouse embryonic stem cells (mESCs). Deletion of SIRT1 results in accumulation of sphingomyelin in mESCs, primarily due to reduction of SMPDL3B, a GPI-anchored plasma membrane bound sphingomyelin phosphodiesterase. Mechanistically, SIRT1 regulates transcription of Smpdl3b through c-Myc. Functionally, SIRT1 deficiency-induced accumulation of sphingomyelin increases membrane fluidity and impairs neural differentiation in vitro and in vivo. Our findings discover a key regulatory mechanism for sphingolipid homeostasis and neural differentiation, further imply that pharmacological manipulation of SIRT1-mediated sphingomyelin degradation might be beneficial for treatment of human neurological diseases.
All cells in the brain start life as stem cells which are yet to have a defined role in the body. A wide range of molecules and chemical signals guide stem cells towards a neuronal fate, including a group of molecules called sphingolipids. These molecules sit in the membrane surrounding the cell and play a pivotal role in a number of processes which help keep the neuronal cell healthy. Various enzymes work together to break down sphingolipids and remove them from the membrane. Defects in these enzymes can result in excess levels of sphingolipids, which can lead to neurodegenerative diseases, such as Alzheimer's, Parkinson's and Huntington's disease. But how these enzymes are used and controlled during neuronal development is still somewhat of a mystery. To help answer this question, Fan et al. studied an enzyme called SIRT1 which has been shown to alleviate symptoms in animal models of neurodegenerative diseases. Stem cells were extracted from a mouse embryo lacking the gene for SIRT1 and cultured in the laboratory. These faulty cells were found to have superfluous amounts of sphingolipids, which made their membranes more fluid and reduced their ability to develop into neuronal cells. Further investigation revealed that SIRT1 regulates the degradation of sphingolipids by promoting the production of another enzyme called SMPDL3B. Fan et al. also found that when female mice were fed a high-fat diet, this caused sphingolipids to accumulate in their embryos which lacked the gene for SIRT1; this, in turn, impaired the neural development of their offspring. These findings suggest that targeting SIRT1 may offer new strategies for treating neurological diseases. The discovery that embryos deficient in SIRT1 are sensitive to high-fat diets implies that activating this enzyme might attenuate some of the neonatal complications associated with maternal obesity.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Neurais/enzimologia , Neurogênese , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuína 1/metabolismo , Esfingolipídeos/metabolismo , Animais , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Sirtuína 1/genética , Transcrição GênicaRESUMO
Pluripotent stem cells (PSCs), such as embryonic stem cells and induced pluripotent stem cells, give rise to all kinds of functional cells, making them promising for successful application in regenerative medicine. However, there is concern that a PSC-derived differentiated cell population may form teratomas when used for cell therapy if the population contains undifferentiated PSCs. Therefore, for the success of regenerative medicine, it is crucial to establish methods that induce complete PSC differentiation and eliminate the contamination of PSCs. Here, I show that the dihydroorotate dehydrogenase (DHODH) inhibitor brequinar (BRQ) induced cell cycle arrest, cell death, and stemness loss in mouse PSCs (mPSCs), whereas it was less toxic against normal tissue-specific stem cells and differentiating cells. I demonstrate that BRQ-pretreated mPSCs did not form teratomas after being transplanted into NOD/SCID mice. Moreover, BRQ administration to teratoma-bearing mice prevented tumor growth and decreased PSC marker levels in the tumor without any visible effects in the differentiated germ layer cells and the mice. Collectively, these data suggested that DHODH inhibitors such as BRQ can be indispensable in the fundamental methods of PSC-based therapy.
Assuntos
Compostos de Bifenilo/farmacologia , Di-Hidro-Orotato Desidrogenase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Células-Tronco Embrionárias Murinas/enzimologia , Animais , Linhagem Celular , Di-Hidro-Orotato Desidrogenase/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Mouse embryonic stem cells (mESCs) are pluripotent cells that possess the ability to self-renew and differentiate into three germ layers. Owing to these characteristics, mESCs act as important models for stem cell research and are being used in many clinical applications. Among the many cathepsins, cathepsin A (Ctsa), a serine protease, affects the function and properties of stem cells. However, studies on the role of Ctsa in stem cells are limited. Here, we observed a significant increase in Ctsa expression during mESC differentiation at protein levels. Furthermore, we established Ctsa knockdown mESCs. Ctsa knockdown led to Erk1/2 phosphorylation, which in turn inhibited the pluripotency of mESCs and induced G2/M cell cycle arrest to inhibit mESC proliferation. The knockdown also induced abnormal differentiation in mESCs and aberrant expression of differentiation markers. Furthermore, we identified inhibition of teratoma formation in nude mice. Our results suggested that Ctsa affects mESC pluripotency, proliferation, cell cycle and differentiation, and highlighted the potential of Ctsa to act as a core factor that can regulate various mESC properties. SIGNIFICANCE OF THE STUDY: Our results indicate that cathepsin A (Ctsa) affects the properties of mESCs. Inhibition of Ctsa resulted in a decrease in the pluripotency of mouse embryonic stem cells (mESCs). Further, Ctsa suppression resulted in decreased proliferation via cell cycle arrest. Moreover, Ctsa inhibition reduced differentiation abilities and formation of teratoma in mESCs. Our results demonstrated that Ctsa is an important factor controlling mESC abilities.
Assuntos
Catepsina A/metabolismo , Diferenciação Celular , Proliferação de Células , Sistema de Sinalização das MAP Quinases , Células-Tronco Embrionárias Murinas/enzimologia , Animais , Catepsina A/genética , Linhagem Celular , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Pontos de Checagem da Fase M do Ciclo Celular/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologiaRESUMO
The identification of novel mechanisms to maintain embryonic stem cell (ESC) pluripotency is of crucial importance, because the currently used culture conditions are not suitable for ESCs from all species. In this study, we show that the protein kinase D (PKD) inhibitor CID755673 (CID) is able to maintain the undifferentiated state of mouse ESCs in combination with the mitogen-activated protein kinase kinase (MEK) inhibitor. The expression levels of PKD members, including PKD1, PKD2 and PKD3, were low in mouse ESCs but significantly increased under differentiation conditions. Therefore, depletion of three PKD genes was able to phenocopy PKD inhibition. Mechanistically, PKD inhibition activated PI3K/AKT signaling by increasing the level of AKT phosphorylation, and the addition of a PI3K/AKT signaling pathway inhibitor partially reduced the cellular response to PKD inhibition. Importantly, the self-renewal-promoting effect of CID was maintained in human ESCs. Simultaneous knockdown of the three human PKD isoforms enabled short-term self-renewal in human ESCs, whereas PI3K/AKT signaling inhibition eliminated this self-renewal ability downstream of the PKD inhibitor. These findings expand our understanding of the gene regulatory network of ESC pluripotency.
Assuntos
Azepinas/farmacologia , Benzofuranos/farmacologia , Células-Tronco Embrionárias Murinas/enzimologia , Proteína Quinase C/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genéticaRESUMO
Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/enzimologia , Mutação , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Biossíntese de Proteínas/genética , RNA Ribossômico 18S/metabolismoRESUMO
Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass of blastocysts, and they exist in different states of pluripotency-naïve and primed states. Pten is a well-known tumor suppressor. Here, we generated Pten-/- mouse ESCs with the CRISPR-Cas9 system and verified that Pten-/- ESCs maintained naïve pluripotency by blocking Gsk3ß activity. Serum/LIF and 2i (MAPK and GSK3 inhibitors) conditions are commonly used for ESC maintenance. We show that the Pten-inhibitor SF1670 contributed to sustaining mouse ESCs and that Pten activation by the S380A, T382A, and T383A mutations (Pten-A3) suppressed the pluripotency of ESCs. The in vivo teratoma formation ability of SF1670-treated ESCs increased, while the Pten-A3 mutations suppressed teratoma formation. Furthermore, the embryoid bodies derived from Pten-deficient ESCs or SF1670-treated wild-type ESCs showed greater expression of ectoderm and pluripotency markers. These results suggest that Pten-mediated Gsk3ß modulates the naïve pluripotency of ESCs and that Pten ablation regulates the lineage-specific differentiation.
Assuntos
Diferenciação Celular , Linhagem da Célula , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Embrionárias Murinas/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Animais , Linhagem Celular , Corpos Embrioides/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/genética , Camundongos , Camundongos Nus , Mutação , PTEN Fosfo-Hidrolase/genética , Fenótipo , Transdução de Sinais , Teratoma/enzimologia , Teratoma/genética , Teratoma/patologiaRESUMO
Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation, and gene expression in DKO ESCs and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, whereas unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild-type (WT) cells but not from 5fC-enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, for example, at promoters, boundaries of chromatin loops, and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to down-regulation of neighboring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions, and DNA sequence that determines differential CTCF binding and regulates gene expression.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Células-Tronco Embrionárias Murinas/enzimologia , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/química , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Elementos Isolantes/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/enzimologia , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Transcriptional regulation occurs via changes to rates of different biochemical steps of transcription, but it remains unclear which rates are subject to change upon biological perturbation. Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release.
Assuntos
Células-Tronco Embrionárias Murinas/enzimologia , RNA Polimerase II/metabolismo , RNA/biossíntese , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Ativação Transcricional , Animais , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Modelos Genéticos , Ligação Proteica , RNA/genética , RNA Polimerase II/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Fatores de TempoRESUMO
Unintended outcomes of cancer therapy include ionizing radiation (IR)-induced stem cell depletion, diminished regenerative capacity, and accelerated aging. Stem cells exhibit attenuated DNA damage response (DDR) and are hypersensitive to IR, as compared to differentiated non-stem cells. We performed genomic discovery research to compare stem cells to differentiated cells, which revealed Phosphoprotein phosphatase 2A (PP2A) as a potential contributor to susceptibility in stem cells. PP2A dephosphorylates pATM, γH2AX, pAkt etc. and is believed to play dual role in regulating DDR and apoptosis. Although studied widely in cancer cells, the role of PP2A in normal stem cell radiosensitivity is unknown. Here we demonstrate that constitutively high expression and radiation induction of PP2A in stem cells plays a role in promoting susceptibility to irradiation. Transient inhibition of PP2A markedly restores DNA repair, inhibits apoptosis, and enhances survival of stem cells, without affecting differentiated non-stem and cancer cells. PP2Ai-mediated stem cell radioprotection was demonstrated in murine embryonic, adult neural, intestinal, and hematopoietic stem cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Oxazóis/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos da radiação , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Masculino , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Embrionárias Murinas/patologia , Células-Tronco Embrionárias Murinas/efeitos da radiação , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/efeitos da radiação , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células-Tronco/enzimologia , Células-Tronco/patologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Although transplantation of stem cells improves recovery of the nervous tissue, little is known about the influence of different brain regions on transplanted cells. After we confirmed that cells with uniform differentiation potential can be generated in independent experiments, one million of neural stem cells isolated from B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse embryos were transplanted into the brain 24â¯h after induction of stroke. The lateral ventricles, the corpus callosum and the striatum were tested. Two and four weeks after the transplantation, the cells transplanted in all three regions have been attracted to the ischemic core. The largest number of attracted cells has been observed after transplantation into the striatum. Their differentiation pattern and expression of neuroligin 1, SynCAM 1, postsynaptic density protein 95 and synapsin 1 followed the same pattern observed during in vitro cultivation and it did not differ among the tested regions. Differentiation pattern of the cells transplanted in the stroke-affected and healthy animals was the same. On the other hand, neural stem cells transplanted in the striatum of the animals affected by stroke exhibited significantly increased survival rates reaching 260⯱â¯19%, when compared to cells transplanted in their wild type controls. Surprisingly, improved survival two and four weeks after transplantation was not due to increased proliferation of the grafted cells and it was accompanied by decreased levels of activity of Casp3 (19.56⯱â¯3.1% in the stroke-affected vs. 30.14⯱â¯2.4% in healthy animals after four weeks). We assume that the decreased levels of Casp3 in cells transplanted near the ischemic region was linked to increased vasculogenesis, synaptogenesis, astrocytosis and axonogenesis detected in the host tissue affected by ischemia.
Assuntos
Caspase 3/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , Acidente Vascular Cerebral/metabolismo , Animais , Movimento Celular/fisiologia , Ventrículos Laterais/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/enzimologia , Neurogênese , Transplante de Células-Tronco/métodosRESUMO
Mouse embryonic stem cells (ESCs) cultured in serum are characterized by hyper-phosphorylated RB protein, lack of G1 control, and rapid progression through the cell cycle. Here, we show that ESCs grown in the presence of two small-molecule inhibitors (2i ESCs) have a longer G1-phase with hypo-phosphorylated RB, implying that they have a functional G1 checkpoint. Deletion of RB, P107, and P130 in 2i ESCs results in a G1-phase similar to that of serum ESCs. Inhibition of the ERK signaling pathway in serum ESCs results in the appearance of hypo-phosphorylated RB and the reinstatement of a G1 checkpoint. In addition, induction of a dormant state by the inhibition of MYC, resembling diapause, requires the presence of the RB family proteins. Collectively, our data show that RB-dependent G1 restriction point signaling is active in mouse ESCs grown in 2i but abrogated in serum by ERK-dependent phosphorylation.
Assuntos
Ciclo Celular , Células-Tronco Pluripotentes/citologia , Animais , Fase G1 , Sistema de Sinalização das MAP Quinases , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Pluripotentes/enzimologia , Proteína do Retinoblastoma/metabolismoRESUMO
BACKGROUND: Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells. OBJECTIVE: In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs. METHODS: A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting. RESULTS: EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium. CONCLUSION: The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Genes myc , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Meios de Cultura , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/enzimologia , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.
Assuntos
Diferenciação Celular , Regulação Enzimológica da Expressão Gênica , Células-Tronco Embrionárias Murinas/enzimologia , Miócitos Cardíacos/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/biossíntese , Animais , Antígenos de Diferenciação/biossíntese , Linhagem Celular , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Miócitos Cardíacos/citologiaRESUMO
Histone deacetylases (HDACs) play crucial roles during mammalian development and for cellular homeostasis. In addition, these enzymes are promising targets for small molecule inhibitors in the treatment of cancer and neurological diseases. Conditional HDAC knock-out mice are excellent tools for defining the functions of individual HDACs in vivo and for identifying the molecular targets of HDAC inhibitors in disease. Here, we describe the generation of tissue-specific HDAC knock-out mice and delineate a strategy for the generation of conditional HDAC knock-in mice.
Assuntos
Blastocisto/enzimologia , Cromatina/metabolismo , Epigênese Genética , Vetores Genéticos/metabolismo , Histona Desacetilase 1/genética , Células-Tronco Embrionárias Murinas/enzimologia , Animais , Blastocisto/citologia , Southern Blotting , Sistemas CRISPR-Cas , Cromatina/química , Cromossomos Artificiais Bacterianos/química , Cromossomos Artificiais Bacterianos/metabolismo , Cruzamentos Genéticos , Feminino , Técnicas de Introdução de Genes , Vetores Genéticos/química , Histona Desacetilase 1/deficiência , Recombinação Homóloga , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Especificidade de Órgãos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The protein sequences of class I HDACs in mice and humans are 96-99 % identical. These highly conserved proteins have crucial roles in biological processes, such as proliferation and development, which is reflected in the lethality that occurs in conventional whole body knockout mice. Therefore, conditional knockouts are inevitable to investigate the functions of class I HDACs in mice. Here, we describe the generation of conditional class I Hdac knockout mice, using Hdac1 as an example. We explain a relatively quick procedure to generate the necessary target vectors by recombination-mediated genetic engineering and gateway techniques. Furthermore, we show how to culture, target, and screen for positively recombined ES cells. Additionally, we present a dual recombination system, which allows the deletion of class I Hdacs at any time by a tamoxifen inducible Cre.
Assuntos
Blastocisto/enzimologia , Epigênese Genética , Vetores Genéticos/metabolismo , Histona Desacetilase 1/genética , Recombinação Homóloga , Células-Tronco Embrionárias Murinas/enzimologia , Animais , Blastocisto/citologia , Southern Blotting , Cruzamentos Genéticos , Feminino , Vetores Genéticos/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Histona Desacetilase 1/deficiência , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno/farmacologiaRESUMO
The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1(+) (Flk-1(+)) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1(+) cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration.