Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 52
Filtrar
1.
Cell ; 163(1): 230-45, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26365490

RESUMO

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.


Assuntos
Células-Tronco Embrionárias/virologia , Retrovirus Endógenos/genética , Provírus/genética , Animais , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Células-Tronco de Carcinoma Embrionário/virologia , Epigênese Genética , Camundongos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
2.
Development ; 149(12)2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35608036

RESUMO

HBXIP, also named LAMTOR5, has been well characterized as a transcriptional co-activator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5, and that depletion of Hbxip compromises the self-renewal of embryonic stem cells (ESCs), with reduced expression of pluripotency genes, reduced cell proliferation and decreased colony-forming capacity. In addition, both Hbxip-/- ESCs and E7.5 embryos displayed defects in ectodermal and mesodermal differentiation. Mechanistically, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, displayed differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/- and p18-/- mice, lacking subunits of the Ragulator complex, also shared similar phenotypes, embryonic lethality and retarded growth around E7-E8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.


Assuntos
Desenvolvimento Embrionário , Células-Tronco Embrionárias , Animais , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Transdução de Sinais
3.
Int J Mol Sci ; 25(5)2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38473939

RESUMO

Embryonic stem cells (ESCs) favor glycolysis over oxidative phosphorylation for energy production, and glycolytic metabolism is critical for pluripotency establishment, maintenance, and exit. However, an understanding of how glycolysis regulates the self-renewal and differentiation of ESCs remains elusive. Here, we demonstrated that protein lactylation, regulated by intracellular lactate, contributes to the self-renewal of ESCs. We further showed that Esrrb, an orphan nuclear receptor involved in pluripotency maintenance and extraembryonic endoderm stem cell (XEN) differentiation, is lactylated on K228 and K232. The lactylation of Esrrb enhances its activity in promoting ESC self-renewal in the absence of the LIF and XEN differentiation of ESCs by increasing its binding at target genes. Our studies reveal the importance of protein lactylation in the self-renewal and XEN differentiation of ESCs, and the underlying mechanism of glycolytic metabolism regulating cell fate choice.


Assuntos
Células-Tronco Embrionárias , Endoderma , Endoderma/metabolismo , Diferenciação Celular/genética
4.
Exp Brain Res ; 241(11-12): 2735-2750, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37845379

RESUMO

Stroke is reported to be the second leading cause of death worldwide, among which ischemic stroke has fourfold greater incidence than intracerebral hemorrhage. Excitotoxicity induced by NMDAR plays a central role in ischemic stroke-induced neuronal death. However, intervention targeted NMDARs against ischemic stroke has failed, which may result from the complex composition of NMDARs and the dynamic changes of their subunits. In this current study, the levels of NR1, NR2A and NR2B subunits of NMDARs were observed upon different time points during the reperfusion after 1 h ischemia with the western blot assay. It was found that the changes of NR1 subunit were only detected after ischemia 1 h/reperfusion 1 day (1 d). While, the changes of NR2A and NR2B subunits may last to ischemia 1 h/reperfusion 7 day(7 d), indicating that NR2subunits may be a potential target for ischemia-reperfusion injuries at the sub-acute stage of ischemic stroke. Simultaneously, mitochondrial injuries in neurons were investigated with transmission electron microscopy (TEM), and mitochondrial dysfunction was evaluated with mitochondrial membrane proteins oxidative respiratory chain complex and OCR. When the antagonist of NMDARs was used before ischemic exposure, the neuronal mitochondrial dysfunction was alleviated, suggesting that these aberrant deviations of NMDARs from basal levels led to mitochondrial dysfunction. Furthermore, when the antagonist of NR2B was administrated intracerebroventricularly at the sub-acute cerebral ischemia, the volume of cerebral infarct region was decreased and the neural functions were improved. To sum up, the ratio of NR2B-containing NMDARs is vital for mitochondrial homeostasis and then neuronal survival. NR2B-targeted intervention should be chosen at the sub-acute stage of cerebral ischemia.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Humanos , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/metabolismo , Infarto Cerebral/metabolismo , AVC Isquêmico/metabolismo , Neurônios/metabolismo
5.
Nucleic Acids Res ; 49(20): 11596-11613, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34723322

RESUMO

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.


Assuntos
Dobramento de RNA , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/metabolismo , Inativação do Cromossomo X , Animais , Autoantígenos/química , Autoantígenos/metabolismo , Sítios de Ligação , Linhagem Celular , Camundongos , Ligação Proteica , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
6.
Proc Natl Acad Sci U S A ; 117(5): 2519-2525, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964807

RESUMO

The highly conserved COP9 signalosome (CSN), composed of 8 subunits (Cops1 to Cops8), has been implicated in pluripotency maintenance of human embryonic stem cells (ESCs). Yet, the mechanism for the CSN to regulate pluripotency remains elusive. We previously showed that Cops2, independent of the CSN, is essential for the pluripotency maintenance of mouse ESCs. In this study, we set out to investigate how Cops5 and Cops8 regulate ESC differentiation and tried to establish Cops5 and Cops8 knockout (KO) ESC lines by CRISPR/Cas9. To our surprise, no Cops5 KO ESC clones were identified out of 127 clones, while three Cops8 KO ESC lines were established out of 70 clones. We then constructed an inducible Cops5 KO ESC line. Cops5 KO leads to decreased expression of the pluripotency marker Nanog, proliferation defect, G2/M cell-cycle arrest, and apoptosis of ESCs. Further analysis revealed dual roles of Cops5 in maintaining genomic stability of ESCs. On one hand, Cops5 suppresses the autophagic degradation of Mtch2 to direct cellular metabolism toward glycolysis and minimize reactive oxygen species (ROS) production, thereby reducing endogenous DNA damage. On the other hand, Cops5 is required for high DNA damage repair (DDR) activities in ESCs. Without Cops5, elevated ROS and reduced DDR activities lead to DNA damage accumulation in ESCs. Subsequently, p53 is activated to trigger G2/M arrest and apoptosis. Altogether, our studies reveal an essential role of Cops5 in maintaining genome integrity and self-renewal of ESCs by regulating cellular metabolism and DDR pathways.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Reparo do DNA , Células-Tronco Embrionárias/enzimologia , Instabilidade Genômica , Peptídeo Hidrolases/metabolismo , Animais , Apoptose , Complexo do Signalossomo COP9/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Técnicas de Inativação de Genes , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosforilação Oxidativa , Peptídeo Hidrolases/genética , Espécies Reativas de Oxigênio/metabolismo
7.
Development ; 144(21): 3957-3967, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28947533

RESUMO

The Hippo pathway modulates the transcriptional activity of Yap to regulate the differentiation of the inner cell mass (ICM) and the trophectoderm (TE) in blastocysts. Yet how Hippo signaling is differentially regulated in ICM and TE cells is poorly understood. Through an inhibitor/activator screen, we have identified Rho as a negative regulator of Hippo in TE cells, and PKA as a positive regulator of Hippo in ICM cells. We further elucidated a novel mechanism by which Rho suppresses Hippo, distinct from the prevailing view that Rho inhibits Hippo signaling through modulating cytoskeleton remodeling and/or cell polarity. Active Rho prevents the phosphorylation of Amot Ser176, thus stabilizing the interaction between Amot and F-actin, and restricting the binding between Amot and Nf2. Moreover, Rho attenuates the interaction between Amot and Nf2 by binding to the coiled-coil domain of Amot. By blocking the association of Nf2 and Amot, Rho suppresses Hippo in TE cells.


Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurofibromina 2/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Angiomotinas , Animais , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Ectoderma/citologia , Ectoderma/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/química , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/química , Modelos Biológicos , Fosforilação , Ligação Proteica , Domínios Proteicos , Quinases Associadas a rho/metabolismo
8.
Nucleic Acids Res ; 46(7): 3468-3486, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29447390

RESUMO

Embryonic stem cells (ESCs) and meiosis are featured by relatively higher frequent homologous recombination associated with DNA double strand breaks (DSB) repair. Here, we show that Pold3 plays important roles in DSB repair, telomere maintenance and genomic stability of both ESCs and spermatocytes in mice. By attempting to generate Pold3 deficient mice using CRISPR/Cas9 or transcription activator-like effector nucleases, we show that complete loss of Pold3 (Pold3-/-) resulted in early embryonic lethality at E6.5. Rapid DNA damage response and massive apoptosis occurred in both outgrowths of Pold3-null (Pold3-/-) blastocysts and Pold3 inducible knockout (iKO) ESCs. While Pold3-/- ESCs were not achievable, Pold3 iKO led to increased DNA damage response, telomere loss and chromosome breaks accompanied by extended S phase. Meanwhile, loss of Pold3 resulted in replicative stress, micronucleation and aneuploidy. Also, DNA repair was impaired in Pold3+/- or Pold3 knockdown ESCs. Moreover, Pold3 mediates DNA replication and repair by regulating 53BP1, RIF1, ATR and ATM pathways. Furthermore, spermatocytes of Pold3 haploinsufficient (Pold3+/-) mice with increasing age displayed impaired DSB repair, telomere shortening and loss, and chromosome breaks, like Pold3 iKO ESCs. These data suggest that Pold3 maintains telomere integrity and genomic stability of both ESCs and meiosis by suppressing replicative stress.


Assuntos
DNA Polimerase III/genética , Replicação do DNA/genética , Células-Tronco Embrionárias/metabolismo , Instabilidade Genômica/genética , Telômero/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Meiose/genética , Camundongos Knockout , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
9.
Proc Natl Acad Sci U S A ; 112(44): E5936-43, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26483458

RESUMO

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC self-renewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs.


Assuntos
Células-Tronco Embrionárias/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Instabilidade Genômica , Transdução de Sinais , Animais , Ciclo Celular , Morte Celular , Células-Tronco Embrionárias/enzimologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Proteína Homeobox Nanog , Telômero
10.
Biochem Biophys Res Commun ; 482(4): 590-596, 2017 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-27864143

RESUMO

The genetic causes for familial nonmedullary thyroid cancer (FNMTC) remain largely unknown. Through genetic linkage analysis and exome sequencing, C14orf93 (RTFC), PYGL, and BMP4 were identified as susceptibility gene candidates in a FNMTC family. By examining the expression and the oncogenic functions of these candidate genes, PYGL and BMP4 were excluded. We further characterized the functions of the uncharacterized gene RTFC in thyroid cancer. RTFC promotes thyroid cancer cell survival under starving conditions, and thyroid cancer cell migration. The R115Q, V205M and G209D RTFC mutants enhance the colony forming capacity of thyroid cancer cells, and are able to transform normal thyroid cells. In summary, our data suggest the roles of RTFC in thyroid carcinogenesis.


Assuntos
Carcinoma/genética , Carcinoma/patologia , Proteínas de Neoplasias/genética , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Sequência de Bases , Carcinoma Papilar , Exoma , Feminino , Ligação Genética , Predisposição Genética para Doença , Humanos , Masculino , Linhagem , Mutação Puntual , Câncer Papilífero da Tireoide , Glândula Tireoide/metabolismo
12.
J Virol ; 88(11): 5956-66, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24623438

RESUMO

UNLABELLED: Phosphatidylinositol 4-phosphate (PI4P) is well known to be upregulated during hepatitis C virus (HCV) replication. The role of PI4 kinases in HCV has been extensively investigated. Whether the PI4P phosphatase Sac1 is altered by HCV remains unclear. Here, we identified ARFGAP1 to be a novel host factor for HCV replication. We further show that Sac1 interacts with ARFGAP1 and inhibits HCV replication. The elevation of PI4P induced by HCV NS5A is abrogated when the coatomer protein I (COPI) pathway is inhibited. We also found an interaction between NS5A and ARFGAP1. Furthermore, we identified a conserved cluster of positively charged amino acids in NS5A critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. Our data demonstrate that ARFGAP1 is a host factor for HCV RNA replication. ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the site of HCV replication to maintain high levels of PI4P. Our findings provide an additional mechanism by which HCV enhances formation of a PI4P-rich environment. IMPORTANCE: PI4P is enriched in the replication area of HCV; however, whether PI4P phosphatase Sac1 is subverted by HCV is not established. The detailed mechanism of how COPI contributes to viral replication remains unknown, though COPI components were hijacked by HCV. We demonstrate that ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the HCV replication area to maintain high-level PI4P generated by NS5A. Furthermore, we identify a conserved cluster of positively charged amino acids in NS5A, which are critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. This study will shed mechanistic insight on how other RNA viruses hijack COPI and Sac1.


Assuntos
Microambiente Celular/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genética , Linhagem Celular , Primers do DNA/genética , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Plasmídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genética
13.
Carcinogenesis ; 35(5): 1144-53, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24464787

RESUMO

Tumor angiogenesis plays an important role in the development of cancer. Previously, we reported that hepatitis B X-interacting protein (HBXIP) functioned as an oncoprotein in breast cancer. However, the role of HBXIP in angiogenesis in breast cancer remains poorly understood. In the present study, we show that the oncoprotein HBXIP plays crucial roles in the event. We observed that the expression levels of HBXIP were positively correlated with those of fibroblast growth factor 8 (FGF8) or vascular endothelial growth factor (VEGF) in clinical breast cancer tissues. Then, we demonstrated that HBXIP was able to upregulate FGF8 through activation of its promoter involving direct binding to cAMP response element-binding protein (CREB) in breast cancer cells and thereby increased its secretion. Strikingly, we identified another pathway that HBXIP upregulated FGF8 and VEGF through inhibiting miRNA-503, which directly targeted 3' untranslated region of FGF8 or VEGF mRNA in the cells. Moreover, we revealed that HBXIP-induced FGF8 could upregulate VEGF expression through activating phosphoinositide 3-kinase (PI3K)/Akt/hypoxia-inducible factor 1-alpha (HIF1α) signaling and increase its secretion. In function, matrigel angiogenesis assay and hemoglobin content analysis uncovered that HBXIP-enhanced FGF8/VEGF boosted tumor angiogenesis and growth in breast cancer in vitro and in vivo in a paracrine/autocrine manner. Thus, we conclude that HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Our finding provides new insights into the mechanism of tumor angiogenesis in breast cancer. Therapeutically, HBXIP may serve as a novel target of tumor angiogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Fator 8 de Crescimento de Fibroblasto/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , MicroRNAs/genética , Neovascularização Patológica/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ativação Transcricional , Carga Tumoral/genética
14.
Dev Biol ; 375(1): 13-22, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23313818

RESUMO

Phosphorylation of Ezrin T567 plays an important role in eight-cell embryo compaction. Yet, it is not clear how Ezrin phosphorylation is regulated during embryo compaction. Here, we demonstrated that inhibition of Mek/Erk or protein kinase C (PKC) signaling reduced the phosphorylation level of Ezrin T567 in eight-cell compacted embryos. Interestingly, the Rho GTPase inhibitor C3-transferase caused basolateral enrichment of atypical PKC (aPKC), as well as basolateral shift of phosphorylated Ezrin, suggesting aPKC may be a key regulator of Ezrin phosphorylation. Moreover, inhibition of PKC, but not Mek/Erk or Rho GTPases, affected the maintenance of Ezrin phosphorylation in compacted embryos. We further identified that aPKC is indeed required for Ezrin phosphorylation in eight-cell embryos. Taken together, Rho GTPases facilitate the apical distribution of aPKC and Ezrin. Subsequently, aPKC and Mek/Erk work together to promote Ezrin phosphorylation at the apical region, which in turn mediates the apical enrichment of filamentous actin, stabilizing the polarized apical region and allowing embryo compaction. Our data also suggested that aPKC might be the Ezrin kinase during eight-cell embryo compaction.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , ADP Ribose Transferases/metabolismo , Actinas/biossíntese , Animais , Toxinas Botulínicas/metabolismo , Células Cultivadas , Técnicas de Cultura Embrionária , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Flavonoides/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores
15.
Acta Biochim Biophys Sin (Shanghai) ; 46(12): 1066-71, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25348736

RESUMO

Ncoa3 is a transcriptional coactivator involved in a wide range of biological processes. Regulation of Ncoa3 protein stability is important to control its activity precisely. Here, we found that deleting amino acid residues 614-740 of Ncoa3 enhances the protein expression level. Replacing two lysine residues, K639 and K673, within this region by arginine, increases the stability of the luciferase fusion protein as well as Ncoa3 protein. When these two lysine residues are mutated to arginine, the overall ubiquitination level of Ncoa3 decreases, indicating that lysine 639 and 673 are its ubiquitination sites. Taken together, we identified two ubiquitination sites at lysine 639 and 673 of Ncoa3. Ubiquitination of these two lysine residues leads to proteasomal degradation of Ncoa3.


Assuntos
Lisina/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Coativador 3 de Receptor Nuclear/química , Estabilidade Proteica , Reação em Cadeia da Polimerase em Tempo Real , Ubiquitinação
16.
Front Immunol ; 15: 1382661, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38558797

RESUMO

Introduction: BTBD8 has been identified as a susceptible gene for inflammatory bowel diseases (IBD). However, the function of BTBD8 in normal development and IBD pathogenesis remains unknown. Methods: We administered drinking water with 3% dextran sodium sulfate (DSS) to wild-type (WT) and Btbd8 knockout (KO) mice for seven consecutive days to induce IBD. Subsequently, we further examined whether Btbd8 KO affects intestinal barrier and inflammation. Results: We demonstrated that Btbd8 deficiency partially protects mice from DSS-induced IBD, even though no obvious phenotypes were observed in Btbd8 KO mice. Btbd8 deletion leads to strengthened tight junctions between intestinal epithelial cells, elevated intestinal stem cell activity, and enhanced mucus layer. All these three mechanisms work together to improve the intestinal barrier integrity in Btbd8 KO mice. In addition, Btbd8 deficiency mitigates inflammation by reducing the expression of IL-1ß and IL-6 by macrophages. Discussion: Our studies validate the crucial role of Btbd8 in IBD pathogenesis, and reveal that Btbd8 deficiency may ameliorate DSS-induced IBD through improving the intestinal barrier integrity, as well as suppressing inflammatory response mediated by macrophages. These findings suggest that Btbd8 could be a promising therapeutic target for the treatment of IBD.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Função da Barreira Intestinal , Colite/induzido quimicamente , Colite/genética , Colite/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Intestinos/patologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia
17.
Int J Biol Sci ; 20(8): 3201-3218, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38904015

RESUMO

Tumor-associated macrophages (TAMs) represent a predominant cellular component within the tumor microenvironment (TME) of pancreatic neuroendocrine neoplasms (pNENs). There is a growing body of evidence highlighting the critical role of exosomes in facilitating communication between tumor cells and TAMs, thereby contributing to the establishment of the premetastatic niche. Nonetheless, the specific mechanisms through which exosomes derived from tumor cells influence macrophage polarization under hypoxic conditions in pNENs, and the manner in which these interactions support cancer metastasis, remain largely unexplored. Recognizing the capacity of exosomes to transfer miRNAs that can modify cellular behaviors, our research identified a significant overexpression of miR-4488 in exosomes derived from hypoxic pNEN cells. Furthermore, we observed that macrophages that absorbed circulating exosomal miR-4488 underwent M2-like polarization. Our investigations revealed that miR-4488 promotes M2-like polarization by directly targeting and suppressing RTN3 in macrophages. This suppression of RTN3 enhances fatty acid oxidation and activates the PI3K/AKT/mTOR signaling pathway through the interaction and downregulation of FABP5. Additionally, M2 polarized macrophages contribute to the formation of the premetastatic niche and advance pNENs metastasis by releasing MMP2, thereby establishing a positive feedback loop involving miR-4488, RTN3, FABP5, and MMP2 in pNEN cells. Together, these findings shed light on the role of exosomal miRNAs from hypoxic pNEN cells in mediating interactions between pNEN cells and intrahepatic macrophages, suggesting that miR-4488 holds potential as a valuable biomarker and therapeutic target for pNENs.


Assuntos
Exossomos , Neoplasias Hepáticas , Macrófagos , MicroRNAs , Tumores Neuroendócrinos , Neoplasias Pancreáticas , MicroRNAs/metabolismo , MicroRNAs/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Exossomos/metabolismo , Humanos , Animais , Camundongos , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/genética , Macrófagos/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/genética , Linhagem Celular Tumoral , Ácidos Graxos/metabolismo , Oxirredução , Microambiente Tumoral , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Camundongos Nus , Transdução de Sinais
18.
J Biol Chem ; 287(45): 38295-304, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22977234

RESUMO

Nuclear receptors, including Esrrb, Dax1, and Nr5a2, have been shown to be involved in pluripotency maintenance. Yet, the role of their coactivators in mouse embryonic stem cells remains unexplored. Here, we demonstrated that the nuclear receptor coactivator 3 (Ncoa3) is essential for pluripotency maintenance. Knockdown of Ncoa3 not only compromises the expression of pluripotency markers but also impairs in vitro and in vivo differentiation potential of mouse ESCs. Ncoa3 binds to the Nanog promoter and recruits the histone acetyltransferase CREB binding protein (CBP) and the histone arginine methyltransferase CARM1 to activate Nanog expression. Moreover, glycogen synthase kinase 3 GSK3 signaling down-regulates the Ncoa3 protein level to suppress Nanog expression. Thus, Ncoa3 not only contributes to self-renewal by activating Nanog but also facilitates ESC differentiation as a break point to disrupt the core transcriptional circuitry of pluripotency.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Coativador 3 de Receptor Nuclear/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Western Blotting , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal , Células NIH 3T3 , Proteína Homeobox Nanog , Coativador 3 de Receptor Nuclear/metabolismo , Gravidez , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Hum Mol Genet ; 20(7): 1339-52, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21239471

RESUMO

Parthenogenetic embryonic stem cells (pESCs) have been generated in several mammalian species from parthenogenetic embryos that would otherwise die around mid-gestation. However, previous reports suggest that pESCs derived from in vivo ovulated (IVO) mature oocytes show limited pluripotency, as evidenced by low chimera production, high tissue preference and especially deficiency in germline competence, a critical test for genetic integrity and pluripotency of ESCs. Here, we report efficient generation of germline-competent pESC lines (named as IVM pESCs) from parthenogenetic embryos developed from immature oocytes of adult mouse ovaries following in vitro maturation (IVM) and artificial activation. In contrast, pESCs derived from IVO oocytes show defective germline competence, consistent with previous reports. Further, IVM pESCs resemble more ESCs from fertilized embryos (fESCs) than do IVO pESCs on genome-wide DNA methylation and global protein profiles. In addition, IVM pESCs express higher levels of Blimp1, Lin28 and Stella, relative to fESCs, and in their embryoid bodies following differentiation. This may indicate differences in differentiation potentially to the germline. The mechanisms for acquisition of pluripotency and germline competency of IVM pESCs from immature oocytes remain to be determined.


Assuntos
Antígenos de Diferenciação/biossíntese , Células-Tronco Embrionárias , Oócitos , Ovário , Partenogênese , Células-Tronco Pluripotentes , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Ovário/citologia , Ovário/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
20.
Proc Natl Acad Sci U S A ; 107(1): 69-74, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20018659

RESUMO

Both p53 and the Wnt signaling pathway play important roles in regulating the differentiation of mouse embryonic stem cells (mESCs). However, it is not known whether they directly and/or functionally crosstalk in mESCs. Here we report a surprising antidifferentiation function of p53 in mESCs through directly regulating the Wnt signaling pathway. A chromatin-immunoprecipitation-based microarray (ChIP-chip) and gene expression microarray assays reveal that the Wnt signaling pathway is significantly (P value, 0.000048) overrepresented in p53-regulated genes in mESCs. The expression of five Wnt ligand genes is robustly induced by various genotoxic and nongenotoxic insults in a p53-dependent manner. Moreover, the induction of these Wnt genes is greatly attenuated in mouse embryonic fibroblast (MEF) cells and ESC-derived neural stem/progenitor cells, suggesting that the induction is mESC specific. It is established that the activation of the Wnt signaling pathway inhibits the differentiation of mESCs. Consistent with this notion, we detected an antidifferentiation activity from the conditioned medium (CM) collected from UV (UV)-treated mESCs. This antidifferentiation activity can be lowered by either the addition of Wnt antagonists into the CM or the reduction of p53 levels in UV-treated mESCs. Therefore, reminiscent of its dual functions on death and survival in somatic cells, p53 appears to regulate both prodifferentiation and antidifferentiation programs in mESCs. Our findings uncover a direct and functional connection between p53 and the Wnt signaling pathway, and expand the catalog of p53 regulated genes in mESCs.


Assuntos
Células-Tronco Embrionárias/fisiologia , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Wnt/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Dano ao DNA , Doxorrubicina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Proteína Homeobox Nanog , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Proteína Supressora de Tumor p53/genética , Proteínas Wnt/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA