RESUMEN

Neural stem and progenitor cells express a variety of receptors that enable them to sense and react to signals emanating from physiological and pathophysiological conditions in the brain as well as elsewhere in the body. Many of these receptors and were first described in investigations of the immune system, particularly with respect to hematopoietic stem cells. This emerging view of neurobiology has two major implications. First, many phenomena known from the hematopoietic system may actually be generalizable to stem cells from many organ systems, reflecting the cells' progenitor-mediated regenerative potential. Second, regenerative interfaces may exist between diverse organ systems; populations of cells of neuroectodermal and hematopoietic origin may interact to play a crucial role in normal brain physiology, pathology, and repair. An understanding of the origins of signals and the neural progenitors' responses might lead to the development of effective therapeutic strategies to counterbalance acute and chronic neurodegenerative processes. Such strategies may include modifying and modulating cells with regenerative potential in subtle ways. For example, stem cells might be able to detect pathology-associated signals and be used as "interpreters" to mediate drug and other therapeutic interventions. This review has focused on the role of inflammation in brain repair. We propose that resident astroglia and blood-born cells both contribute to an inflammatory signature that is unique to each kind of neuronal degeneration or injury. These cells play a key role in coordinating the neural progenitor cell response to brain injury by exerting direct and indirect environmentally mediated influence on neural progenitor cells. We suggest that investigations of the neural progenitor-immunologic interface will provide valuable data related to the mechanisms by which endogenous and exogenous neural progenitor cells react to brain pathology, ultimately aiding in the design of more effective therapeutic applications of stem cell biology. Such improvements will include: (1) ascertaining the proper timing for implanting exogenous neural progenitor cells in relation to the administration of anti-inflammatory agents; (2) identifying what types of molecules might be administered during injury to enhance the mobilization and differentiation of endogenous and exogenous neural progenitor cells while also inhibiting the detrimental aspects of the inflammatory reaction; (3) divining clues as to which molecules may be required to change the lesioned environment in order to invite the homing of reparative neural progenitor cells.

Asunto(s)

Sistema Inmunológico , Sistema Nervioso/patología , Animales , Encéfalo/patología , Diferenciación Celular , Linaje de la Célula , Humanos , Inflamación , Modelos Biológicos , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Células Madre/citología

RESUMEN

The transcription factor PU.1 (also known as Spi-1) plays a critical role in the development of the myeloid lineages, and myeloid cells derived from PU.1(-/-) animals are blocked at the earliest stage of myeloid differentiation. Expression of the PU.1 gene is tightly regulated during normal hematopoietic development, and dysregulation of PU.1 expression can lead to erythroleukemia. However, relatively little is known about how the PU.1 gene is regulated in vivo. Here it is shown that myeloid cell type-specific expression of PU.1 in stable cell lines and transgenic animals is conferred by a 91-kilobase (kb) murine genomic DNA fragment that consists of the entire PU.1 gene (20 kb) plus approximately 35 kb of upstream and downstream sequences, respectively. To further map the important transcriptional regulatory elements, deoxyribonuclease I hypersensitive site mapping studies revealed at least 3 clusters in the PU.1 gene. A 3.5-kb fragment containing one of these deoxyribonuclease I hypersensitive sites, located -14 kb 5' of the transcriptional start site, conferred myeloid cell type-specific expression in stably transfected cell lines, suggesting that within this region is an element important for myeloid specific expression of PU.1. Further analysis of this myeloid-specific regulatory element will provide insight into the regulation of this key transcriptional regulator and may be useful as a tool for targeting expression to the myeloid lineage.

Asunto(s)

Regulación de la Expresión Génica/genética , Proteínas Proto-Oncogénicas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/genética , Animales , Células Cultivadas/citología , Células Cultivadas/metabolismo , Desoxirribonucleasa I/metabolismo , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Transgénicos , Células Mieloides/citología , Células Mieloides/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Linfocitos T/metabolismo , Transactivadores/biosíntesis , Transfección , Células U937/citología , Células U937/metabolismo

RESUMEN

Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.

Asunto(s)

Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Encéfalo/citología , Neuronas/citología , Trasplante de Células Madre , Células Madre/citología , Animales , Antígenos/análisis , Biomarcadores/análisis , Células de la Médula Ósea/fisiología , Diferenciación Celular , Movimiento Celular , Femenino , Técnicas para Inmunoenzimas , Proteínas de Filamentos Intermediarios/análisis , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Nestina , Neuronas/química , Neuronas/inmunología , Fosfopiruvato Hidratasa/análisis , Células Madre/química , Cromosoma Y

RESUMEN

Apoptosis is one of the key tools used by an embryo to regulate cell numbers and sculpt body shape. Although massive numbers of cells die during development, they are so rapidly phagocytosed that very few corpses are ever seen in most embryonic tissues. In this paper, we focus on the catastrophic cell death that occurs as the developing footplate is remodelled to transform webbed regions into free interdigital spaces. In the wild-type embryo, these dead cells are rapidly engulfed and cleared by macrophages. We show that in a macrophageless mouse embryo, null for the haemopoetic-lineage-specific transcription factor, PU.1, the task of phagocytosis is taken over by 'stand-in' mesenchymal neighbours in a clear example of cell redundancy. However, it takes three times as many of these mesenchymal phagocytes to complete the task and, at each stage of the clearance process - in the recognition of apoptotic debris, its engulfment and finally its digestion - they appear to be less efficient than macrophages. A molecular explanation for this may be that several of the engulfment genes expressed by macrophages, including the ABC1 transporter (believed to be part of the phagocytic machinery conserved from Caenorhabditis elegans to mouse), are not upregulated by these 'stand-in' phagocytes.

Asunto(s)

Apoptosis/fisiología , Pie/embriología , Mesodermo/citología , Mesodermo/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo , Fagocitos/fisiología , Fagocitosis , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Transactivadores/genética

RESUMEN

To elucidate the molecular mechanisms involved in the action of common carcinogens, which can act as important cofactors in modulating hepatitis B virus-mediated hepatocellular carcinogenesis, we have investigated the influence of aflatoxin B(1) (AFB), a potent liver carcinogen, as well as benzo[a]pyrene (BP) and 4-aminobiphenyl (4-ABP), carcinogens in cigarette smoke, on the induction of various transcription factors in human hepatoblastoma HepG2 cells. DNA electrophoretic mobility shift assays were performed with nuclear extracts from HepG2 cells treated with 10 micromol/L AFB, 40 micromol/L BP, or 300 micromol/L 4-ABP for 6 and 24 hours. Eight- and 6-fold increases in nuclear transcription factor kappaB (NF-kappaB), and 5- and 10-fold increases in activated protein (AP-1) transcription factor were observed with 24 hours AFB and BP treatments, respectively, whereas 4-ABP treatment resulted in an approximately 4-fold induction of both NF-kappaB and AP-1. Moreover, 4-ABP gave the strongest NF-kappaB activation in 6 hours of treatment. Four- and 10-fold activation of stress protein was detected by a consensus heat shock factor (HSF) sequence binding probe, with AFB and BP treatments, respectively. DNA adducts were observed by immunoassays in HepG2 cells treated with AFB and BP but not with 4-ABP. Increased human hepatitis B virus (HBV) surface antigen (HBsAg) synthesis was detected in AFB- and BP-treated HepG2 cells following transfection with recircularized HBV DNA. These data suggest that certain carcinogen-induced transcription factors may influence viral carcinogenesis and initiate hepatocellular carcinomas (HCC).

Asunto(s)

Carcinógenos/toxicidad , Virus de la Hepatitis B/efectos de los fármacos , Hepatoblastoma/etiología , Neoplasias Hepáticas/etiología , Factores de Transcripción/fisiología , Activación Viral/efectos de los fármacos , Antígenos de Superficie de la Hepatitis B/biosíntesis , Virus de la Hepatitis B/fisiología , Hepatoblastoma/metabolismo , Hepatoblastoma/virología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virología , FN-kappa B/fisiología , Factor de Transcripción AP-1/fisiología , Células Tumorales Cultivadas

RESUMEN

PU.1 is a transcription factor shown to regulate the expression of many important genes in myeloid and B cells. At birth, mice homozygous for the disruption of the PU.1 gene have erythrocytes, megakaryocytes, and T cells, but no mature myeloid or B cells. Cells with an inability to develop to maturity were found in this mouse for B cells, neutrophils, eosinophils, mast cells, and monocytes. Rescue of early monocytic cells by transfection with the PU.1 gene results in renewed development to macrophages. These results demonstrate that PU.1 is an important regulator in the development of cells in the hematopoietic system.

Asunto(s)

Linaje de la Célula/fisiología , Células Madre Hematopoyéticas/citología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Ratones , Monocitos/citología , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética

RESUMEN

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis. Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophage-like cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway.

Asunto(s)

Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica , Lectinas Tipo C , Macrófagos/citología , Lectinas de Unión a Manosa , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Factores de Transcripción , Animales , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/genética , Desarrollo Embrionario y Fetal/fisiología , Antígeno de Macrófago-1/genética , Macrófagos/fisiología , Receptor de Manosa , Ratones , Factor de Transcripción Asociado a Microftalmía , ARN Mensajero/análisis , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptores de Superficie Celular/genética

RESUMEN

Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1-/- myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1-/- colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1-/- fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF-dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1-/- progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.

Asunto(s)

Células Madre Hematopoyéticas/fisiología , Monocitos/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Animales Recién Nacidos , Ensayo de Unidades Formadoras de Colonias , Cruzamientos Genéticos , Feto , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Monocitos/citología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Transactivadores/deficiencia , Transactivadores/genética

RESUMEN

We have cloned a new member of the interferon (IFN)-induced guanylate-binding protein (GBP) family of GTPases, murine GBP-2 (mGBP-2), from bone marrow-derived macrophages. mGBP-2 is located on murine chromosome 3, where it is linked to mGBP-1. With the identification of mGBP-2 there are now two human and two murine GBPs. Like other GBPs, mGBP-2 RNA and protein are induced by IFN-gamma. In addition, mGBP-2 shares with the other GBPs important structural features that distinguish this family from other GTPases. First, mGBP-2 contains only two of the three consensus sequences for nucleotide binding found within the classic GTP binding regions of other GTPases. A second amino acid motif found in mGBP-2 is a potential C-terminal site for isoprenoid modification, called a CaaX sequence. mGBP-2 is prenylated, as detected by [3H]mevalonate incorporation, when expressed in COS cells and preferentially incorporates the C-20 isoprenoid geranylgeraniol. Surprisingly, despite having a functional CaaX sequence, mGBP-2 is primarily cytosolic. GBP proteins are very abundant in IFN-exposed cells, but little is known about their function. mGBP-2 is expressed by IFN-gamma-treated cells from C57Bl/6 mice, whereas mGBP-1 is not. Thus, the identification of mGBP-2 makes possible the study of GBP function in the absence of a second family member.

Asunto(s)

GTP Fosfohidrolasas/aislamiento & purificación , Proteínas de Unión al GTP/genética , Interferón gamma/farmacología , Macrófagos/enzimología , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Células COS , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , Inducción Enzimática , GTP Fosfohidrolasas/sangre , Proteínas de Unión al GTP/biosíntesis , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Prenilación de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding receptors for M-CSF, G-CSF and GM-CSF, were expressed at low levels in PU.1(-/-) fetal liver compared with wild type. To identify additional myeloid genes regulated by PU.1 and C/EBPalpha, we performed representational difference analysis (RDA), a PCR-based subtractive hybridization using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. By introducing a new modification of RDA, that of tissue-specific gene suppression, we could selectively identify a set of differentially expressed genes specific to myeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. In addition, eight novel genes were identified that were upregulated in expression during myeloid differentiation. These methods provide a general strategy for elucidating the genes affected in murine knockout models.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptores del Factor Estimulante de Colonias/genética , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular Transformada , Clonación Molecular/métodos , Gránulos Citoplasmáticos/genética , Proteínas de Unión al ADN/genética , Feto , Genes/genética , Leucopoyesis/genética , Hígado , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/análisis , Transactivadores/genética

RESUMEN

The ets family transcription factor PU.1 is expressed in monocytes/macrophages, neutrophils, mast cells, B cells, and early erythroblasts, but not in T cells. We have recently shown that PU.1 gene disruption results in mice with no detectable monocytes/macrophages and B cells but T-cell development is retained. Although neutrophil development occurred in these mice, it was delayed and markedly reduced. We now proceed to demonstrate that PU. 1 null hematopoietic cells fail to proliferate or form colonies in response to macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF), and granulocyte/macrophage CSF (GM-CSF). In contrast, PU.1 null cells did proliferate and form colonies in response to interleukin-3 (IL-3), although the response was reduced as compared with control littermates. Compared with control cells, PU.1 null cells had minimal expression of G- and GM-CSF receptors and no detectable M-CSF receptors. The size of individual myeloid colonies produced from PU.1 null primitive and committed myeloid progenitors in the presence of IL-3, IL-6, and stem cell factor (SCF) were reduced compared with controls. Under these conditions, PU.1 null progenitors produced neutrophils but not monocytes/macrophages. These observations suggest that PU.1 gene disruption induces additional cell-autonomous effects that are independent of the alterations in myeloid growth factor receptor expression. Our results demonstrate that PU.1 gene disruption affects a number of developmentally regulated hematopoietic processes that can, at least in part, explain the changes in myeloid development and reduction in myeloid and neutrophil expansion observed in PU.1 null mice.

Asunto(s)

Hematopoyesis/genética , Células Madre Hematopoyéticas/patología , Síndromes de Inmunodeficiencia/patología , Proteínas Proto-Oncogénicas/deficiencia , Transactivadores/deficiencia , Animales , Diferenciación Celular/genética , División Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Resistencia a Medicamentos , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Síndromes de Inmunodeficiencia/genética , Leucopenia/genética , Leucopenia/patología , Subgrupos Linfocitarios/patología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Neutrófilos/patología , Fenotipo , Proteínas Proto-Oncogénicas/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/deficiencia , Receptores de Factor Estimulante de Colonias de Granulocito/deficiencia , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/deficiencia , Transactivadores/genética

RESUMEN

Members of the Ets family of transcription factors mediate transcriptional responses of multiple signaling pathways in diverse cell types and organisms. Targeted deletion of the conserved DNA binding domain of the Ets2 transcription factor results in the retardation and death of homozygous mouse embryos before 8.5 days of embryonic development. Defects in extraembryonic tissue gene expression and function include deficient expression of matrix metalloproteinase-9 (MMP-9, gelatinase B), persistent extracellular matrix, and failure of ectoplacental cone proliferation. Mutant embryos were rescued by aggregation with tetraploid mouse embryos, which complement the developmental defects by providing functional extraembryonic tissues. Rescued Ets2-deficient mice are viable and fertile but have wavy hair, curly whiskers, and abnormal hair follicle shape and arrangement, resembling mice with mutations of the EGF receptor or its ligands. However, these mice are not deficient in the production of TGFalpha or the EGF receptor. Homozygous mutant cell lines respond mitogenically to TGFalpha, EGF, FGF1, and FGF2. However, FGF fails to induce MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in the Ets2-deficient fibroblasts. Ectopic expression of Ets2 in the deficient fibroblasts restores expression of both matrix metalloproteinases. Therefore, Ets2 is essential for placental function, mediating growth factor signaling to key target genes including MMP-3, MMP-9, and MMP-13 in different cell types, and for regulating hair development.

Asunto(s)

Proteínas de Unión al ADN , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción , Trofoblastos/fisiología , Animales , Secuencia de Bases , Sitios de Unión/genética , Quimera , Cartilla de ADN/genética , Desarrollo Embrionario y Fetal/genética , Desarrollo Embrionario y Fetal/fisiología , Receptores ErbB/genética , Receptores ErbB/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Cabello/anomalías , Masculino , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Fenotipo , Poliploidía , Proteína Proto-Oncogénica c-ets-2 , ARN/genética , ARN/metabolismo , Transducción de Señal

RESUMEN

Osteoclasts are multinucleated cells and the principal resorptive cells of bone. Although osteoclasts are of myeloid origin, the role of haematopoietic transcription factors in osteoclastogenesis has not been explored. Here we show that messenger RNA for the myeloid- and B-cell-specific transcription factor PU.1 progressively increases as marrow macrophages assume the osteoclast phenotype in vitro. The association between PU.1 and osteoclast differentiation was confirmed by demonstrating that PU.1 expression increased with the induction of osteoclastogenesis by either 1,25-dihydroxyvitamin D3 or dexamethasone. Consistent with the participation of PU.1 in osteoclastogenesis, we found that the development of both osteoclasts and macrophages is arrested in PU.1-deficient mice. Reflecting the absence of osteoclasts, PU.1-/- mice exhibit the classic hallmarks of osteopetrosis, a family of sclerotic bone diseases. These animals were rescued by marrow transplantation, with complete restoration of osteoclast and macrophage differentiation, verifying that the PU.1 lesion is intrinsic to haematopoietic cells. The absence of both osteoclasts and macrophages in PU.1-mutant animals suggests that the transcription factor regulates the initial stages of myeloid differentiation, and that its absence represents the earliest developmental osteopetrotic mutant yet described.

Asunto(s)

Osteopetrosis/etiología , Proteínas Proto-Oncogénicas/deficiencia , Transactivadores/deficiencia , Animales , Animales Recién Nacidos , Médula Ósea/patología , Trasplante de Médula Ósea , Resorción Ósea , Huesos/patología , Diferenciación Celular , Línea Celular , Eliminación de Gen , Hematopoyesis/fisiología , Macrófagos/patología , Ratones , Ratones Transgénicos , Mutación , Osteoclastos/patología , Osteoclastos/fisiología , Osteopetrosis/genética , Osteopetrosis/patología , Osteopetrosis/terapia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , ARN Mensajero/biosíntesis , Células del Estroma/patología , Transactivadores/genética , Transactivadores/fisiología

RESUMEN

Transcription factors play an important role choreographing lineage commitment and expansion of blood cells. Nuclear factors that are expressed primarily or exclusively in hematopoietic cells are likely candidates for regulating blood cell development. The transcription factor PU.1 is found only in hematopoietic cells, whereas ets-2, a related family member, is ubiquitously expressed. To compare the role of these two transcription factors in macrophage development, embryonic stem (ES) cells with a homozygous disruption of either the PU.1 or the ets-2 gene were generated. The ability of both knockout ES cells to differentiate to macrophages was tested. Normal development of macrophages, as determined by histochemical and immunohistochemical analysis, from PU.1 knockout ES cells was significantly blocked. Furthermore, the expression of known markers associated with macrophages, such as c-fms, CD11b, CD18 and granulocyte-macrophage colony-stimulating factor receptor, were not detected by reverse transcriptase-polymerase chain reaction. In contrast to the PU.1 knockout ES cells, macrophages, development from the ets-2 knockout ES cells was normal. Although both PU.1 and ets-2 are found in macrophages, these data show a distinct role for the lineage-restricted PU.1 transcription factor in macrophage development.

Asunto(s)

Proteínas de Unión al ADN , Macrófagos/citología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras , Células Madre/citología , Transactivadores/fisiología , Factores de Transcripción , Animales , Antígenos CD18/análisis , Diferenciación Celular , Células L , Antígeno de Macrófago-1/análisis , Ratones , Proteína Proto-Oncogénica c-ets-2 , Receptor de Factor Estimulante de Colonias de Macrófagos/análisis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Transfección

RESUMEN

PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3-5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic-treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

Asunto(s)

Hematopoyesis/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores , Animales , Linfocitos B/citología , Sitios de Unión , Diferenciación Celular , ADN/metabolismo , Citometría de Flujo , Macrófagos/citología , Ratones , Neutrófilos/citología , Proteínas Proto-Oncogénicas/fisiología , Linfocitos T/citología

RESUMEN

Transcription factors belonging to the ets family regulate gene expression and share a conserved ETS DNA-binding domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The domain is similar to alpha+beta ("winged") helix-turn-helix DNA-binding proteins. The crystal structure of the PU.1 ETS domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ets proteins confirms this complex as a paradigm for ets DNA recognition. The second helix in the helix-turn-helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.

Asunto(s)

Proteínas de Unión al ADN/química , ADN/química , Secuencias Hélice-Giro-Hélice , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Análisis Mutacional de ADN , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas c-ets , Homología de Secuencia de Aminoácido , Transactivadores/química , Factores de Transcripción/química

RESUMEN

The Y box is a conserved sequence in the promoter of major histocompatibility complex (MHC) class II genes, which contains a CCAAT sequence (CCAAT box). Previously, we partially purified the DNA-binding protein that recognizes the Y box of the I-A beta gene and showed that it consisted of two components (factors A and B) both of which were necessary for optimal DNA binding. The genes for the heteromeric protein NF-Y (NF-YA and NF-YB), which binds to the I-E alpha Y box have been cloned. We subsequently isolated the genes for NF-YA and NF-YB using oligonucleotides designed from the published sequences. NF-YA and NF-YB were tested for binding to the I-A beta and I-E alpha Y boxes. While neither NF-YA or NF-YB alone bound to the Y box, when the components were mixed the complex bound to the I-A beta Y box with high affinity. Moreover, NF-YA and NF-YB could be complemented for binding to DNA by factor B or factor A, respectively. These results suggest that the active binding protein is NF-YA in factor A extracts and NF-YB in factor B extracts. Finally, antibodies against NF-YA and NF-YB were shown to induce a supershift when nuclear extracts were added to the double-stranded oligodeoxynucleotide covering the Y box of the I-A beta gene. Antisense expression constructs of both NF-YA and NF-YB were made and their effect on expression from the I-A beta promoter was tested. Either antisense construction, when transfected into cells, lowered the expression of a reporter gene linked to the I-A beta promoter. This study provides direct evidence of the identification of NF-YA and NF-YB as the previously described factors A and B. Moreover, these results strongly implicate NF-Y in the expression of the MHC class II gene I-A beta.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Genes MHC Clase II , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , ADN , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Unión Proteica

RESUMEN

PU.1 is a tissue-specific transcription factor that is expressed in cells of the hematopoietic lineage including macrophages, granulocytes, and B lymphocytes. Bone marrow-derived macrophages transfected with an antisense PU.1 expression construct or treated with antisense oligonucleotides showed a decrease in proliferation compared with controls. In contrast, bone marrow macrophages transfected with a sense PU.1 expression construct displayed enhanced macrophage colony-stimulating factor (M-CSF)-dependent proliferation. Interestingly, there was no effect of sense or antisense constructs of PU.1 on the proliferation of the M-CSF-independent cell line, suggesting that the response was M-CSF dependent. This was further supported by the finding that macrophages transfected with a sense or an antisense PU.1 construct showed, respectively, an increased or a reduced level of surface expression of receptors for M-CSF. The enhancement of proliferation seems to be selective for PU.1, since transfections with several other members of the ets family, including ets-2 and fli-1, had no effect. Various mutants of PU.1 were also tested for their ability to affect macrophage proliferation. A reduction in macrophage proliferation was found when cells were transfected with a construct in which the DNA-binding domain of PU.1 was expressed. The PEST (proline-, glutamic acid-, serine-, and threonine-rich region) sequence of the PU.1 protein, which is an important domain for protein-protein interactions in B cells, was found to have no influence on PU.1-enhanced macrophage proliferation when an expression construct containing PU.1 minus the PEST domain was transfected into bone marrow-derived macrophages. In vivo, PU.1 is phosphorylated on several serine residues. The transfection of plasmids containing PU.1 with mutations at each of five serines showed that only positions 41 and 45 are critical for enhanced macrophage proliferation. We conclude that PU.1 is necessary for the M-CSF-dependent proliferation of macrophages. One of the proliferation-relevant targets of this transcription factor could be the M-CSF receptor.

Asunto(s)

Activación de Macrófagos , Macrófagos/citología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores , Animales , Secuencia de Bases , Células de la Médula Ósea , ADN sin Sentido/química , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular

RESUMEN

The Ets family of transcription factors, of which there are now about 35 members regulate gene expression during growth and development. They share a conserved domain of around 85 amino acids which binds as a monomer to the DNA sequence 5'-C/AGGAA/T-3'. We have determined the crystal structure of an ETS domain complexed with DNA, at 2.3-A resolution. The domain is similar to alpha + beta (winged) 'helix-turn-helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8 degrees. The domain contacts the DNA by a novel loop-helix-loop architecture. Four of amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix-turn-helix' motif, which binds downstream and on the opposite strand.

Asunto(s)

Proteínas de Unión al ADN/química , ADN/química , Secuencias Hélice-Giro-Hélice , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Proteínas Oncogénicas de Retroviridae , Homología de Secuencia de Aminoácido

RESUMEN

Class II genes of the MHC show a striking homology upstream of the transcription start site that is composed of three conserved sequences (S, X and Y boxes, each separated by 15-20 bp). The presence of the S-box sequence in the mouse MHC class II gene I-A Beta was examined for its influence on the expression of this gene. Deletion or mutation of the S box decreased the induction of chloramphenicol acetyltransferase (CAT) activity in B lymphocytes by 32%. In macrophages, deletion or mutation of the S box abolished interferon-gamma (IFN-gamma) inducibility of CAT activity. Using a gel-retardation assay, we have identified a nuclear factor whose binding site overlaps the 7-mer conserved sequence of the S box. This factor is present in lymphocytes, macrophages, mastocytes and fibroblasts. Surprisingly, binding of this nuclear factor to DNA was induced by IFN-gamma in bone-marrow-derived macrophages, but not in macrophage-like cell lines. The binding site for this factor was defined by DNase I footprinting and partially purified by using an affinity column containing double-stranded oligonucleotides containing a sequence of the S box. A prominent protein of 43 kDa was found that bound specifically to the S-box sequence.

Asunto(s)

ADN/genética , ADN/metabolismo , Genes MHC Clase II , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , Secuencia Conservada , Elementos de Facilitación Genéticos , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis , Regiones Promotoras Genéticas , Factores de Transcripción/química , Transfección