RESUMO
Neural stem cells (NSCs) in the adult rat subventricular zone (SVZ) generate new striatal neurons during several months after ischemic stroke. Whether the microglial response associated with ischemic injury extends into SVZ and influences neuroblast production is unknown. Here, we demonstrate increased numbers of activated microglia in ipsilateral SVZ concomitant with neuroblast migration into the striatum at 2, 6, and 16 weeks, with maximum at 6 weeks, following 2 h middle cerebral artery occlusion in rats. In the peri-infarct striatum, numbers of activated microglia peaked already at 2 weeks and declined thereafter. Microglia in SVZ were resident or originated from bone marrow, with maximum proliferation during the first 2 weeks postinsult. In SVZ, microglia exhibited ramified or intermediate morphology, signifying a downregulated inflammatory profile, whereas amoeboid or round phagocytic microglia were frequent in the peri-infarct striatum. Numbers of microglia expressing markers of antigen-presenting cells (MHC-II, CD86) increased in SVZ but very few lymphocytes were detected. Using quantitative PCR, strong short- and long-term increase (at 1 and 6 weeks postinfarct) of insulin-like growth factor-1 (IGF-1) gene expression was detected in SVZ tissue. Elevated numbers of IGF-1-expressing microglia were found in SVZ at 2, 6, and 16 weeks after stroke. At 16 weeks, 5% of microglia but no other cells in SVZ expressed the IGF-1 protein, which mitigates apoptosis and promotes proliferation and differentiation of NSCs. The long-term accumulation of microglia with proneurogenic phenotype in the SVZ implies a supportive role of these cells for the continuous neurogenesis after stroke.
Assuntos
Ventrículos Laterais/fisiopatologia , Microglia/fisiologia , Neurogênese/fisiologia , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/cirurgia , Análise de Variância , Animais , Células Apresentadoras de Antígenos/metabolismo , Transplante de Medula Óssea/métodos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/efeitos da radiação , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Ectodisplasinas/genética , Ectodisplasinas/metabolismo , Lateralidade Funcional , Proteínas de Fluorescência Verde/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Antígenos Comuns de Leucócito/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos , Microglia/metabolismo , Fenótipo , Ratos , Ratos Wistar , Acidente Vascular Cerebral/fisiopatologia , Fatores de TempoRESUMO
During brain embryogenesis, transcription factors drive stem cells towards neuronal fate. Here we show that the transcription factor Pax6 increased in vitro generation of neurons from striatal but not cortical neural stem cells (NSCs), derived from 6 to 9 weeks old human fetuses, without affecting survival and proliferation. Overexpression of mouse Pax6 produced increased numbers of GABA+ and DARPP-32+ (characteristic of striatum) but not glutamate+ neurons (characteristic of cortex). Pax6-overexpressing cells survived and migrated to the same extent as control cells at 1 month after intrastriatal transplantation into newborn rats and generated more neuroblasts. Overexpression of mouse Pax6 in human NSCs also leads to altered levels of lineage-appropriate genes as revealed by Q-PCR. Our data suggest that Pax6 function is conserved between species since its overexpression activates similar genes in mouse and human NSCs. Also, that Pax6 overexpression in striatal NSCs increases the number of neurons but their region-specificity is maintained.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Fatores de Transcrição Box Pareados/fisiologia , Proteínas Repressoras/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/embriologia , Corpo Estriado/metabolismo , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transplante de Células-Tronco/métodosRESUMO
Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPARgamma2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPARgamma2 induce adipocyte differentiation with comparable kinetics and efficiency.
Assuntos
Adipogenia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , PPAR gama/genética , Transativadores/genética , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Análise de Variância , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Cinética , Ligantes , Camundongos , Células NIH 3T3 , PPAR gama/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/metabolismoRESUMO
Reversible contraction of immunoglobulin loci juxtaposes the variable (V) genes next to the (diversity)-joining-constant ((D)JC) gene domain, thus facilitating V-(D)J recombination. Here we show that the T cell receptor beta (Tcrb) and T cell receptor alphadelta (Tcra-Tcrd) loci also underwent long-range interactions by looping in double-negative and double-positive thymocytes, respectively. Contraction of the Tcrb and Tcra loci occurred in rearranging thymocytes and was reversed at the next developmental stage. Decontraction of the Tcrb locus probably prevented further V(beta)-DJ(beta) rearrangements in double-positive thymocytes by separating the V(beta) genes from the DJC(beta) domain. In most double-negative cells, one Tcrb allele was recruited to pericentromeric heterochromatin. Such allelic positioning may facilitate asynchronous V(beta)-DJ(beta) recombination. Hence, pericentromeric recruitment and locus 'decontraction' seem to contribute to the initiation and maintenance of allelic exclusion at the Tcrb locus.
Assuntos
Rearranjo Gênico do Linfócito T/imunologia , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/imunologia , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/imunologia , Regiões Constantes de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Hibridização in Situ Fluorescente , Linfopoese/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Fator de Transcrição PAX5/imunologiaRESUMO
The development of mature B lymphoid cells involves a highly orchestrated regulation of stage- and lineage-specific genes. In this study, we report an analysis of the human surrogate L chain VpreB promoter. The promoter has an overall homology of 56% to the mouse counterpart and displays a preB cell-restricted activity in transient transfections in cell lines. The promoter harbors three independent binding sites for early B cell factor (EBF) as defined by EMSA and supershift experiments. These sites were important for the full function of the promoter in a preB cell line, and chromatin immunoprecipitation experiments indicate that EBF interacts with the promoter in vivo. In addition to this, ectopic expression of EBF induces the activity of a reporter gene under control of the VpreB promoter in epithelioid HeLa cells, an effect augmented by coexpression of the basic-helix-loop helix transcription factor E47. The ability to interact directly with E47 was shared by the promoters controlling the human mb-1 and B29 genes. These data indicate that the human VpreB promoter is a direct target for activation by EBF and E47 and that functional collaboration between these proteins may be of great importance in human B cell development.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Região Variável de Imunoglobulina/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Regiões Promotoras Genéticas/imunologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Transformada , Rearranjo Gênico de Cadeia Leve de Linfócito B , Células HeLa , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Cadeias Leves de Imunoglobulina , Cadeias Leves Substitutas da Imunoglobulina , Células Jurkat , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de TranscriçãoRESUMO
The early B cell factor (EBF) is a transcription factor shown crucial for the development of B lymphocytes. The protein is expressed from the earliest stages of B cell development until the mature B cell stage, but the control elements responsible for the regulation of the gene are unknown. In this study, we report of the identification of a promoter region flanking the EBF gene. Several transcription start sites were identified by primer extension analysis in a region approximately 3.1 kb from the predicted ATG. Transient transfections revealed that this region was able to stimulate transcription of a reporter gene in B lymphoid and to a lesser extent, myeloid cells, but not in a pre-T cell line. The promoter was also able to functionally interact with E47, suggesting that the EBF gene may be a direct target for activation by E-proteins. In addition, functional binding of EBF to its own promoter was confirmed by EMSA and transfection assays indicating that the EBF protein may be involved in an autoregulatory loop. Finally, a tissue-restricted factor was able to bind an upstream regulatory region in B-lineage cells, further supporting the idea that the cloned promoter participates in the regulation of stage and lineage specific expression of the EBF gene.
Assuntos
Clonagem Molecular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Sequências Hélice-Alça-Hélice/genética , Sequências Hélice-Alça-Hélice/imunologia , Regiões Promotoras Genéticas/imunologia , Transativadores/biossíntese , Transativadores/genética , Fatores de Transcrição/fisiologia , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/imunologia , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Composição de Bases , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Elementos E-Box/genética , Elementos E-Box/imunologia , Éxons/genética , Éxons/imunologia , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Fatores de Transcrição TCF , Transativadores/química , Transativadores/fisiologia , Proteína 1 Semelhante ao Fator 7 de TranscriçãoRESUMO
B lymphocyte development is a complex biological process critically dependent on the transcription factor early B cell factor (EBF). To deepen understanding of the roles for EBF in this process, we have used Pearson correlation analysis to evaluate microarray data from a set of mouse B lymphoid cell lines representing different stages of development. Comparing the expression pattern of EBF to that of the other genes in the data set revealed that VpreB1, mb-1, and lambda5, all known target genes, presented high correlation values to EBF. High correlations were also seen for the VpreB3 and CD19 genes and biochemical as well as functional data supported that they are target genes for EBF even though the expression of CD19 was critically dependent of Pax-5. We also obtained evidence for extensive collaborative actions of EBF and E47 even though microarray analysis of hematopoetic progenitor cells ectopically expressing these proteins suggested that they activated only a subset of pre-B cell restricted genes.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Transativadores/genética , Animais , Antígenos CD19/biossíntese , Sítios de Ligação , Células da Medula Óssea/citologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Dimerização , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Oligonucleotídeos/química , Fator de Transcrição PAX5 , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatística como Assunto , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The childhood malignancy neuroblastoma is derived from developmentally arrested sympathetic nervous system precursor cells. To obtain further insight into the molecular processes involved in the formation of these tumors, we decided to investigate the functional role of Olf/EBF (O/E) transcription factors in human neuroblastoma cells. We here report that O/E-1 and O/E-2 are expressed at variable levels in neuroblastoma cell lines and that O/E proteins could be identified by electrophoretic mobility shift assays. To identify potential neuronal target genes for O/E proteins in neuroblastoma cells we investigated the ability of a set of neuronal promoters to interact with O/E-1 in electrophoretic mobility shift assays. This analysis suggested that the Chromogranin A (CgA) and SCG10 promoters both contained binding sites for O/E-1. O/E-1 was able to activate the CgA promoter in vivo and mutation of the O/E-1 binding site in the CgA promoter reduced the functional activity of the element to about 60% of the wild-type in neuroblastoma cells, supporting the idea that O/E proteins may be involved in the control of the CgA promoter. Furthermore, overexpression of O/E-1 in hippocampal progenitor cells led to neurite outgrowth, indicative of a role for O/E proteins in neuronal differentiation.