RESUMO
The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC's long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a "mixed" neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the "neuronal" genes are bound by both SOX2 and AP1.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Genoma , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição AP-1/genética , Animais , Sequência de Bases , Diferenciação Celular/genética , Regulação para Baixo/genética , Deleção de Genes , Lentivirus/metabolismo , Camundongos , Modelos Biológicos , Neuroglia/metabolismo , Neurônios/metabolismo , RNA-Seq , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSCs). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Furthermore, pharmacological inhibition by T-5224 of FOS/JUN AP1 complex binding to its targets decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its re-expression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 re-expression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; furthermore, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.
Assuntos
Células-Tronco Neurais , Proteínas Proto-Oncogênicas c-fos , Fatores de Transcrição SOXB1 , Animais , Proliferação de Células/genética , Autorrenovação Celular/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismoRESUMO
The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of ß-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and ß039 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the ß/γ+ß globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the ß-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.
Assuntos
Receptores Nucleares Órfãos , gama-Globinas , Fator II de Transcrição COUP/genética , Fator II de Transcrição COUP/metabolismo , Proteínas de Transporte/genética , Humanos , Regiões Promotoras Genéticas , gama-Globinas/genéticaRESUMO
Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain gliomas, and is essential to maintain CSC. In mouse high-grade glioma pHGG cells in culture, Sox2 deletion causes cell proliferation arrest and inability to reform tumors after transplantation in vivo; in Sox2-deleted cells, 134 genes are derepressed. To identify genes mediating Sox2 deletion effects, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Ebf1, Hey2, Zfp423, and Cdkn2b, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis of each gene, individually or in combination (Ebf1 + Cdkn2b), significantly antagonized the proliferation arrest caused by Sox2 deletion. The same genes also repressed clonogenicity in primary human glioblastoma-derived CSC-like lines. These experiments identify a network of critical tumor suppressive Sox2-targets whose inhibition by Sox2 is involved in glioma CSC maintenance, defining new potential therapeutic targets.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Oligodendroglioma , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias Encefálicas/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação para Baixo , Glioma/genética , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas Repressoras , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , TransativadoresRESUMO
Visual system development involves the formation of neuronal projections connecting the retina to the thalamic dorso-lateral geniculate nucleus (dLGN) and the thalamus to the visual cerebral cortex. Patients carrying mutations in the SOX2 transcription factor gene present severe visual defects, thought to be linked to SOX2 functions in the retina. We show that Sox2 is strongly expressed in mouse postmitotic thalamic projection neurons. Cre-mediated deletion of Sox2 in these neurons causes reduction of the dLGN, abnormal distribution of retino-thalamic and thalamo-cortical projections, and secondary defects in cortical patterning. Reduced expression, in mutants, of Sox2 target genes encoding ephrin-A5 and the serotonin transport molecules SERT and vMAT2 (important for establishment of thalamic connectivity) likely provides a molecular contribution to these defects. These findings unveil thalamic SOX2 function as a novel regulator of visual system development and a plausible additional cause of brain-linked genetic blindness in humans.
RESUMO
The SOX2 transcription factor is critical for neural stem cell (NSC) maintenance and brain development. Through chromatin immunoprecipitation (ChIP) and chromatin interaction analysis (ChIA-PET), we determined genome-wide SOX2-bound regions and Pol II-mediated long-range chromatin interactions in brain-derived NSCs. SOX2-bound DNA was highly enriched in distal chromatin regions interacting with promoters and carrying epigenetic enhancer marks. Sox2 deletion caused widespread reduction of Pol II-mediated long-range interactions and decreased gene expression. Genes showing reduced expression in Sox2-deleted cells were significantly enriched in interactions between promoters and SOX2-bound distal enhancers. Expression of one such gene, Suppressor of Cytokine Signaling 3 (Socs3), rescued the self-renewal defect of Sox2-ablated NSCs. Our work identifies SOX2 as a major regulator of gene expression through connections to the enhancer network in NSCs. Through the definition of such a connectivity network, our study shows the way to the identification of genes and enhancers involved in NSC maintenance and neurodevelopmental disorders.
Assuntos
Cromatina/metabolismo , Células-Tronco Neurais/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Células Cultivadas , Redes Reguladoras de Genes/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Fatores de Transcrição SOXB1/deficiência , Fatores de Transcrição SOXB1/genética , Peixe-ZebraRESUMO
Sox2 is a transcription factor active in the nervous system, within different cell types, ranging from radial glia neural stem cells to a few specific types of differentiated glia and neurons. Mutations in the human SOX2 transcription factor gene cause various central nervous system (CNS) abnormalities, involving hippocampus and eye defects, as well as ataxia. Conditional Sox2 mutation in mouse, with different Cre transgenes, previously recapitulated different essential features of the disease, such as hippocampus and eye defects. In the cerebellum, Sox2 is active from early embryogenesis in the neural progenitors of the cerebellar primordium; Sox2 expression is maintained, postnatally, within Bergmann glia (BG), a differentiated cell type essential for Purkinje neurons functionality and correct motor control. By performing Sox2 Cre-mediated ablation in the developing and postnatal mouse cerebellum, we reproduced ataxia features. Embryonic Sox2 deletion (with Wnt1Cre) leads to reduction of the cerebellar vermis, known to be commonly related to ataxia, preceded by deregulation of Otx2 and Gbx2, critical regulators of vermis development. Postnatally, BG is progressively disorganized, mislocalized, and reduced in mutants. Sox2 postnatal deletion, specifically induced in glia (with GLAST-CreERT2), reproduces the BG defect, and causes (milder) ataxic features. Our results define a role for Sox2 in cerebellar function and development, and identify a functional requirement for Sox2 within postnatal BG, of potential relevance for ataxia in mouse mutants, and in human patients.
Assuntos
Ataxia/metabolismo , Vermis Cerebelar/crescimento & desenvolvimento , Vermis Cerebelar/metabolismo , Neuroglia/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Animais Recém-Nascidos , Ataxia/patologia , Células Cultivadas , Vermis Cerebelar/patologia , Regulação da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos Transgênicos , Mutação , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neuroglia/patologia , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição SOXB1/genética , Transmissão Sináptica/fisiologiaRESUMO
The identification of drugs capable of reactivating γ-globin to ameliorate ß-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/ß globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing ß-globin (ß-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and ß-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of ß-hemoglobinopathies.
Assuntos
Globinas beta/análise , gama-Globinas/análise , Anemia Falciforme/metabolismo , Ácido Butírico/metabolismo , Hemoglobina Fetal/metabolismo , Humanos , Hidroxiureia/metabolismo , Células K562 , Globinas beta/metabolismo , Talassemia beta/metabolismo , gama-Globinas/metabolismoRESUMO
The stem cell-determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor-induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Células-Tronco Neoplásicas/fisiologia , Oligodendroglioma/metabolismo , Fatores de Transcrição SOXB1/fisiologia , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Vacinas Anticâncer , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodendroglioma/imunologia , Oligodendroglioma/patologia , Oligodendroglioma/terapia , Fragmentos de Peptídeos/imunologia , Fatores de Transcrição SOXB1/imunologia , Transcriptoma , Células Tumorais CultivadasRESUMO
The transcription factor GATA1 is essential for erythroid and megakaryocytic cell differentiation. Gata1 hematopoietic regulatory domain (G1HRD) has been shown to recapitulate endogenous Gata1 gene expression in transgenic mouse assays in vivo. G1HRD contains a promoter-proximal enhancer composed of a GATA-palindrome motif, four CP2-binding sites and two CACCC boxes. We prepared transgenic reporter mouse lines in which green fluorescent protein and ß-galactosidase expression are driven by wild-type G1HRD (as a positive control) and the G1HRD harboring mutations within these cis-acting elements (as the experimental conditions), respectively. Exploiting this transgenic dual reporter (TDR) assay, we show here that in definitive erythropoiesis, G1HRD activity was markedly affected by individual mutations in the GATA-palindrome motif and the CACCC boxes. Mutation of CP2-binding sites also moderately decreased G1HRD activity. The combined mutation of the CP2-binding sites and the GATA-palindrome motif resulted in complete loss of G1HRD activity. In contrast, in primitive erythroid cells, individual mutations of each element did not affect G1HRD activity; G1HRD activity was abolished only when these three mutations were combined. These results thus show that all three elements independently and cooperatively contribute to G1HRD activity in vivo in definitive erythropoiesis, although these are contributing redundantly to primitive erythropoiesis.
Assuntos
Elementos Facilitadores Genéticos , Fator de Transcrição GATA1/genética , Regiões Promotoras Genéticas , Animais , Sítios de Ligação , Diferenciação Celular , Eritropoese , Fator de Transcrição GATA1/metabolismo , Genes Reporter , Camundongos , Camundongos Transgênicos , Sequências Reguladoras de Ácido NucleicoRESUMO
The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.
Assuntos
Células-Tronco Adultas/transplante , Insuficiência Cardíaca/terapia , Miócitos Cardíacos/citologia , Células-Tronco Adultas/metabolismo , Animais , Células da Medula Óssea/metabolismo , Proteínas de Fluorescência Verde/análise , Coração/fisiologia , Insuficiência Cardíaca/induzido quimicamente , Humanos , Isoproterenol , Masculino , Camundongos , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Fator de Células-Tronco/metabolismoRESUMO
The Sox2 transcription factor is active in stem/progenitor cells throughout the developing vertebrate central nervous system. However, its conditional deletion at E12.5 in mouse causes few brain developmental problems, with the exception of the postnatal loss of the hippocampal radial glia stem cells and the dentate gyrus. We deleted Sox2 at E9.5 in the telencephalon, using a Bf1-Cre transgene. We observed embryonic brain defects that were particularly severe in the ventral, as opposed to the dorsal, telencephalon. Important tissue loss, including the medial ganglionic eminence (MGE), was detected at E12.5, causing the subsequent impairment of MGE-derived neurons. The defect was preceded by loss of expression of the essential ventral determinants Nkx2.1 and Shh, and accompanied by ventral spread of dorsal markers. This phenotype is reminiscent of that of mice mutant for the transcription factor Nkx2.1 or for the Shh receptor Smo. Nkx2.1 is known to mediate the initial activation of ventral telencephalic Shh expression. A partial rescue of the normal phenotype at E14.5 was obtained by administration of a Shh agonist. Experiments in Medaka fish indicate that expression of Nkx2.1 is regulated by Sox2 in this species also. We propose that Sox2 contributes to Nkx2.1 expression in early mouse development, thus participating in the region-specific activation of Shh, thereby mediating ventral telencephalic patterning induction.
Assuntos
Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Proteínas Nucleares/genética , Fatores de Transcrição SOXB1/fisiologia , Telencéfalo/embriologia , Fatores de Transcrição/genética , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Gravidez , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Telencéfalo/metabolismo , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo , Ativação Transcricional/genéticaRESUMO
How Kit expression is regulated in the germline remains unknown. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, are involved in spermatogonia and oocyte differentiation. In the male, deletion of each factor causes loss of Kit-expressing spermatogonia in the prepuberal testis. In the female, SOHLH1 and SOHLH2 ablations cause oocyte loss in the neonatal ovary. To investigate whether Kit expression is regulated by these two factors in male germ cells, we examined SOHLH1 and SOHLH2 expression during fetal and postnatal mouse development. We found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal at Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with all-trans retinoic acid. We found that E-box sequences within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing Sohlh1 or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box-containing probe. In vivo co-immunoprecipitations indicated that the two proteins interact and overexpression of both factors increases endogenous Kit expression in embryonic stem cells. SOHLH1 was found by ChIP analysis to occupy an E-box-containing region within the Kit promoter in spermatogonia chromatin. Our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signaling involved in spermatogonia differentiation and spermatogenetic progression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/genética , Espermatogônias/fisiologia , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Espermatogônias/citologiaRESUMO
RATIONALE: Embryonic and fetal myocardial growth is characterized by a dramatic increase in myocyte number, but whether the expansion of the myocyte compartment is dictated by activation and commitment of resident cardiac stem cells (CSCs), division of immature myocytes or both is currently unknown. OBJECTIVE: In this study, we tested whether prenatal cardiac development is controlled by activation and differentiation of CSCs and whether division of c-kit-positive CSCs in the mouse heart is triggered by spontaneous Ca(2+) oscillations. METHODS AND RESULTS: We report that embryonic-fetal c-kit-positive CSCs are self-renewing, clonogenic and multipotent in vitro and in vivo. The growth and commitment of c-kit-positive CSCs is responsible for the generation of the myocyte progeny of the developing heart. The close correspondence between values computed by mathematical modeling and direct measurements of myocyte number at E9, E14, E19 and 1 day after birth strongly suggests that the organogenesis of the embryonic heart is dependent on a hierarchical model of cell differentiation regulated by resident CSCs. The growth promoting effects of c-kit-positive CSCs are triggered by spontaneous oscillations in intracellular Ca(2+), mediated by IP3 receptor activation, which condition asymmetrical stem cell division and myocyte lineage specification. CONCLUSIONS: Myocyte formation derived from CSC differentiation is the major determinant of cardiac growth during development. Division of c-kit-positive CSCs in the mouse is promoted by spontaneous Ca(2+) spikes, which dictate the pattern of stem cell replication and the generation of a myocyte progeny at all phases of prenatal life and up to one day after birth.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Coração/embriologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , Técnicas de Cultura Embrionária , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Modelos Teóricos , Organogênese/fisiologia , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
BACKGROUND: During late differentiation, erythroid cells undergo profound changes involving actin filament remodeling. One of the proteins controlling actin dynamics is gelsolin, a calcium-activated actin filament severing and capping protein. Gelsolin-null (Gsn(-/-)) mice generated in a C57BL/6 background are viable and fertile.1 DESIGN AND METHODS: We analyzed the functional roles of gelsolin in erythropoiesis by: (i) evaluating gelsolin expression in murine fetal liver cells at different stages of erythroid differentiation (using reverse transcription polymerase chain reaction analysis and immunohistochemistry), and (ii) characterizing embryonic and adult erythropoiesis in Gsn(-/-) BALB/c mice (morphology and erythroid cultures). RESULTS: In the context of a BALB/c background, the Gsn(-/-) mutation causes embryonic death. Gsn(-/-) embryos show defective erythroid maturation with persistence of circulating nucleated cells. The few Gsn(-/-) mice reaching adulthood fail to recover from phenylhydrazine-induced acute anemia, revealing an impaired response to stress erythropoiesis. In in vitro differentiation assays, E13.5 fetal liver Gsn(-/-) cells failed to undergo terminal maturation, a defect partially rescued by Cytochalasin D, and mimicked by administration of Jasplakinolide to the wild-type control samples. CONCLUSIONS: In BALB/c mice, gelsolin deficiency alters the equilibrium between erythrocyte actin polymerization and depolymerization, causing impaired terminal maturation. We suggest a non-redundant role for gelsolin in terminal erythroid differentiation, possibly contributing to the Gsn(-/-) mice lethality observed in mid-gestation.
Assuntos
Células-Tronco Embrionárias/patologia , Eritrócitos/patologia , Eritropoese/genética , Gelsolina/genética , Fígado/patologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Anemia/induzido quimicamente , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Depsipeptídeos/farmacologia , Embrião de Mamíferos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feto , Gelsolina/deficiência , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fenil-Hidrazinas/toxicidadeRESUMO
RATIONALE: Understanding the mechanisms that regulate trafficking of human cardiac stem cells (hCSCs) may lead to development of new therapeutic approaches for the failing heart. OBJECTIVE: We tested whether the motility of hCSCs in immunosuppressed infarcted animals is controlled by the guidance system that involves the interaction of Eph receptors with ephrin ligands. METHODS AND RESULTS: Within the cardiac niches, cardiomyocytes expressed preferentially the ephrin A1 ligand, whereas hCSCs possessed the EphA2 receptor. Treatment of hCSCs with ephrin A1 resulted in the rapid internalization of the ephrin A1-EphA2 complex, posttranslational modifications of Src kinases, and morphological changes consistent with the acquisition of a motile cell phenotype. Ephrin A1 enhanced the motility of hCSCs in vitro, and their migration in vivo following acute myocardial infarction. At 2 weeks after infarction, the volume of the regenerated myocardium was 2-fold larger in animals injected with ephrin A1-activated hCSCs than in animals receiving control hCSCs; this difference was dictated by a greater number of newly formed cardiomyocytes and coronary vessels. The increased recovery in myocardial mass with ephrin A1-treated hCSCs was characterized by further restoration of cardiac function and by a reduction in arrhythmic events. CONCLUSIONS: Ephrin A1 promotes the motility of EphA2-positive hCSCs, facilitates their migration to the area of damage, and enhances cardiac repair. Thus, in situ stimulation of resident hCSCs with ephrin A1 or their ex vivo activation before myocardial delivery improves cell targeting to sites of injury, possibly providing a novel strategy for the management of the diseased heart.
Assuntos
Efrina-A1/genética , Efrina-A2/genética , Células-Tronco Hematopoéticas/citologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Animais , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Citoplasma/metabolismo , Efrina-A1/metabolismo , Efrina-A2/metabolismo , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Ratos , Ratos Endogâmicos F344 , Taquicardia Ventricular/patologia , Taquicardia Ventricular/fisiopatologia , Taquicardia Ventricular/terapiaRESUMO
Sox6 belongs to the Sry (sex-determining region Y)-related high-mobility-group-box family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector.
Assuntos
Células Precursoras Eritroides/fisiologia , Eritropoese/genética , Fatores de Transcrição SOXD/fisiologia , Animais , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Processos de Crescimento Celular/genética , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/metabolismo , Eritropoese/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Células K562 , Camundongos , Modelos Biológicos , Fatores de Transcrição SOXD/genética , Fatores de Transcrição SOXD/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/fisiologia , TransfecçãoRESUMO
The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation.