RESUMEN
Perivascular macrophages (pvMs) are associated with cerebral vasculature and mediate brain drainage and immune regulation. Here, using reporter mouse models, whole brain and section immunofluorescence, flow cytometry, and single cell RNA sequencing, besides the Lyve1+F4/80+CD206+CX3CR1+ pvMs, we identify a CX3CR1- pvM population that shares phagocytic functions and location. Furthermore, the brain parenchyma vasculature mostly hosts Lyve1+MHCII- pvMs with low to intermediate CD45 expression. Using the double Cx3cr1GFP x Cx3cr1-Cre;RosatdT reporter mice for finer mapping of the lineages, we establish that CD45lowCX3CR1- pvMs are derived from CX3CR1+ precursors and require PU.1 during their ontogeny. In parallel, results from the Cxcr4-CreErt2;Rosa26tdT lineage tracing model support a bone marrow-independent replenishment of all Lyve1+ pvMs in the adult mouse brain. Lastly, flow cytometry and 3D immunofluorescence analysis uncover increased percentage of pvMs following photothrombotic induced stroke. Our results thus show that the parenchymal pvM population is more heterogenous than previously described, and includes a CD45low and CX3CR1- pvM population.
Asunto(s)
Macrófagos , Fagocitos , Animales , Ratones , Recuento de Leucocitos , Citometría de Flujo , EncéfaloRESUMEN
The bZip transcription factor MafB is expressed specifically in the myeloid lineage of the hematopoietic system and is up-regulated successively during myeloid differentiation from multipotent progenitors to macrophages. Here we report that this induction reflects an essential role of MafB in early myeloid and monocytic differentiation. We observed that the expression of MafB in transformed chicken hematopoietic precursors dramatically increases the proportion of myeloid colony formation at the expense of multipotent progenitor-type colonies. In addition, the overexpression of MafB in transformed myeloblasts stimulates the rapid formation of macrophages, as judged by morphology, surface marker expression and functional criteria. MafB-induced macrophages exhibit typical levels of phagocytic activity and nitric oxide release after activation by lipopolysaccharide. By contrast, overexpression of the myeloid transcription factor PU.1 in these cells does not induce macrophage differentiation. Furthermore, a dominant-negative allele of MafB inhibits both myeloid colony formation and the differentiation of myeloblasts into macrophages. Taken together, our results indicate that MafB induction is a specific and essential determinant of the monocytic program in hematopoietic cells.
Asunto(s)
Proteínas Aviares , Diferenciación Celular , Proteínas de Unión al ADN , Macrófagos/citología , Monocitos/citología , Proteínas Oncogénicas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Biomarcadores/análisis , Línea Celular Transformada , Tamaño de la Célula , Embrión de Pollo , Ensayo de Unidades Formadoras de Colonias , Genes Dominantes/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Factor de Transcripción MafB , Monocitos/metabolismo , Mutación/genética , Óxido Nítrico/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas v-myb/genética , Proteínas Oncogénicas v-myb/fisiología , Fagocitosis , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Codorniz , Temperatura , Transactivadores/genética , Activación Transcripcional , TransfecciónRESUMEN
Recent studies have shown that hematopoietic transcription factors can engage in multiple protein-protein interactions. Accumulating evidence indicates that specific complexes define differentiation lineages and differentiation stages. It is proposed that these complexes acquire new functions during blood cell differentiation through successive changes in composition - much as discussion topics of groups at a cocktail party take new directions as new people join and others leave.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Factores de Transcripción/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Eritropoyesis/genética , Eritropoyesis/fisiología , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Modelos BiológicosRESUMEN
The EOS47 antigen is an early and specific marker of eosinophil differentiation in the chicken haematopoietic system. To elucidate the transciptional events controlling commitment to the eosinophil lineage, we studied the regulation of the eosinophil-specific EOS47 promoter. This promoter is TATA-less, and binds trancription factors of the Ets, C/EBP, GATA and Myb families. These sites are contained within a 309 bp promoter fragment which is sufficient for specific high level transcription in an eosinophil cell line. Co-transfection experiments in Q2bn fibroblasts showed cooperative activation of the EOS47 proximal promoter by c-Myb, Ets-1/Fli-1, GATA-1 and C/EBPalpha. The Ets-1/Fli-1 and C/EBPalpha proteins were the most potent activators, and acted with high synergy through juxtaposed binding sites located approximately 60 bp upstream of the transcription start site. The Ets-1 and C/EBPalpha proteins were found to associate physically via their DNA-binding domains and to bind their combined binding site cooperatively. GATA-1 showed biphasic regulation of the EOS47 promoter, activating at low and repressing at high protein concentrations. These results demonstrate combinatorial activation of an eosinophil-specific promoter by ubiquitous and lineage-restricted haematopoietic transcription factors. They also indicate that direct interactions between C/EBPs and specific Ets family members, together with GATA-1, are important for eosinophil lineage determination.
Asunto(s)
Antígenos de Superficie/genética , Biomarcadores , Proteínas de Unión al ADN/metabolismo , Eosinófilos/metabolismo , Glicoproteínas de Membrana/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Antígenos de Superficie/metabolismo , Proteínas Aviares , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular , Pollos , ADN/metabolismo , ADN Complementario , Proteínas de Unión al ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Proteínas Proto-Oncogénicas c-myb , Proteínas Recombinantes de Fusión/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genéticaRESUMEN
The distal enhancer region of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) is known to be essential for HIV replication and to contain immediately adjacent E-box and Ets binding sites. Based on a yeast one-hybrid screen we have identified the E-box binding protein USF-1 as a direct interaction partner of Ets-1 and found that the complex acts on this enhancer element. The binding surfaces of USF-1 and Ets-1 map to their DNA-binding domains and although these domains are highly conserved, the interaction is very selective within the respective protein family. USF-1 and Ets-1 synergize in specific DNA binding as well as in the transactivation of reporter constructs containing the enhancer element, and mutations of the individual binding sites dramatically reduce reporter activity in T cells. In addition, a dominant negative Ets-1 mutant inhibits both USF-1-mediated transactivation and the activity of the HIV-1 LTR in T cells. The inhibition is independent of Ets DNA-binding sites but requires the Ets binding surface on USF-1, highlighting the importance of the direct protein-protein interaction. Together these results indicate that the interaction between Ets-1 and USF-1 is required for full transcriptional activity of the HIV-1 LTR in T cells.
Asunto(s)
Realizador del VIH/genética , VIH-1/genética , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/virología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Clonación Molecular , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Dominantes , VIH-1/fisiología , Secuencias Hélice-Asa-Hélice , Humanos , Células Jurkat , Leucina Zippers , Datos de Secuencia Molecular , Mutación , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas c-ets , Codorniz , Proteínas Recombinantes de Fusión , Factores de Transcripción/genética , Activación Transcripcional/fisiología , Transfección , Factores Estimuladores hacia 5'RESUMEN
Using a yeast interaction screen with a DNA-bound Ets-1 protein we have identified MafB as a direct interaction partner. This distant AP-1 relative, which is specifically expressed in myelomonocytic cells, binds to the DNA binding domain of Ets-1 via its basic region/leucine zipper domain. MafB represses Ets-1 transactivation of synthetic promoters containing Ets binding sites in yeast as well as avian cells. Both Ets-1 and Maf family proteins have been implicated in erythroid specific gene expression. Here we show that MafB inhibits Ets-1-mediated transactivation of the transferrin receptor, which is essential for heme synthesis and erythroid differentiation. Consequently, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits the cells' potential to differentiate into erythrocytes, without affecting cellular proliferation.
Asunto(s)
Proteínas Aviares , Proteínas de Unión al ADN , Eritroblastos/citología , Eritrocitos/citología , Eritropoyesis/fisiología , Células Madre Hematopoyéticas/citología , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Eritroblastos/fisiología , Eritrocitos/fisiología , Células Madre Hematopoyéticas/fisiología , Hemo/biosíntesis , Leucina Zippers , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas c-ets , Receptores de Transferrina/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae , Activación Transcripcional , TransfecciónRESUMEN
Using a yeast one-hybrid screen with a DNA-bound Ets-1 protein, we have identified MafB, an AP-1 like protein, as a direct interaction partner. MafB is specifically expressed in myelomonocytic cells and binds to the DNA-binding domain of Ets-1 via its basic region or leucine-zipper domain. Furthermore, it represses Ets-1 transactivation of synthetic promoters containing Ets binding sites and inhibits Ets-1-mediated transactivation of the transferrin receptor, which is known to be essential for erythroid differentiation. Accordingly, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits differentiation without affecting cell proliferation. These results highlight the importance of inhibitory interactions between transcription factors in regulating lineage-specific gene expression.
Asunto(s)
Proteínas Aviares , Proteínas de Unión al ADN/genética , Eritroblastos/citología , Proteínas Oncogénicas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Embrión de Pollo , Células Clonales/fisiología , ADN Complementario/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Eritroblastos/fisiología , Factores de Unión a la G-Box , Regulación de la Expresión Génica/fisiología , Pruebas Genéticas , Hematopoyesis/genética , Macrófagos/fisiología , Factor de Transcripción MafB , Datos de Secuencia Molecular , Proteínas Oncogénicas/metabolismo , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ets , Receptores de Transferrina/genética , Proteínas Represoras/genética , Transactivadores/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Vertebrados , Levaduras/genéticaRESUMEN
From clinical, chemical carcinogenesis and transgenic animal studies, it is evident that wounding has a tumor-promoting effect. We discuss the role of TGF-beta (with special emphasis on TGF-beta 1) in this process and suggest that stromal alterations during wound healing, induced by TGF-beta, can be an important determinant of tumor growth. A tumor and a wound both require similar stromal microenvironments. Thus, a chemically initiated or an oncogene-expressing cell could be complemented to grow into a tumor if it finds itself in a hospitable wound-healing stroma.
Asunto(s)
Transformación Celular Neoplásica/genética , Cocarcinogénesis , Neoplasias/fisiopatología , Factor de Crecimiento Transformador beta/fisiología , Cicatrización de Heridas/fisiología , Animales , Humanos , Ratones , Factor de Crecimiento Transformador beta/genéticaRESUMEN
In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-beta is present locally shortly after wounding, but not unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor-beta 1 (TGF-beta 1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-beta resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still led to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in wound healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-alpha had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-beta release during the wound-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system.
Asunto(s)
Sarcoma Aviar/patología , Factores de Crecimiento Transformadores/farmacología , Heridas y Lesiones/patología , Animales , Anticuerpos , Pollos , Factor de Crecimiento Epidérmico/farmacología , Humanos , Técnicas para Inmunoenzimas , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Recombinantes/farmacología , Sarcoma Aviar/complicaciones , Porcinos , Factores de Crecimiento Transformadores/análisis , Cicatrización de Heridas , Heridas y Lesiones/complicacionesRESUMEN
v-src is an effective carcinogen when expressed from Rous sarcoma virus (RSV) in vivo. Whereas RSV tumors require sustained oncogene expression, their growth is largely a balance between viral recruitment of tissues and host immune destruction of infected cells. We have therefore examined the tumorigenic potential of v-src in the absence of viral recruitment and viral antigen expression. v-src was introduced with high efficiency into chicken wing web tissues using replication-defective (rd) retroviral vectors. Clonal sarcomas were induced rapidly, and, furthermore, v-src potentiated metastatic progression in approximately 0.1%-1% of tumor clones with unexpectedly short latency. rd vectors proved effective not only in transducing v-src into tissues but also as insertional markers of tumor clonality. The rd vector present in most primary and metastatic tumors was a highly truncated form of RSV derived by viral transmission of spliced v-src mRNA; this vector should thus avoid viral recruitment and host anti-viral immune reaction through its complete lack of viral structural genes. Under such conditions v-src maintains strong carcinogenicity in vivo when restricted to clonal tumor growth and can confer rapid metastatic potential on a discrete subset of tumor clones.
Asunto(s)
Virus del Sarcoma Aviar/genética , Virus Defectuosos/genética , Expresión Génica , Oncogenes , Sarcoma Aviar/genética , Transducción Genética , Animales , Secuencia de Bases , Pollos , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Virus Defectuosos/fisiología , Vectores Genéticos , Metástasis de la Neoplasia , Técnicas de Amplificación de Ácido Nucleico , Proteína Oncogénica pp60(v-src)/análisis , Empalme del ARN/genética , ARN Mensajero/genética , Mapeo Restrictivo , Sarcoma Aviar/patología , Replicación ViralRESUMEN
Chickens given injections of Rous sarcoma virus form sarcomas at the site of inoculation (primary tumor) and at the site of experimentally introduced wounds (wound tumor). This latter finding provides a model system to study systematically the mechanisms underlying the cocarcinogenic effects of wounding. Our experiments show the following. (a) Chickens inoculated with a Rous sarcoma virus-derived, replication-defective virus construct fail to elaborate wound tumors in spite of aggressively growing primary tumors. We thus rule out metastasis as a mechanism and conclude that infectious virus is required for wound tumor formation; (b) using bromodeoxyuridine incorporation and immunofluorescence on frozen sections we demonstrate proliferation in the unwounded wing in cell types which are normally targets for Rous sarcoma virus infection and transformation and conclude that proliferation per se is not sufficient to induce wound tumors; (c) using immunohistochemistry for the viral protein p19gag we show that wounding induces virus expression in fibroblasts of newly forming granulation tissue 2 days after injury. We also demonstrate expression of viral mRNA in wound tumors by in situ hybridization with a v-src probe. We discuss the possibility of activation of integrated, silent virus or the preferential infection of a special target cell population as a result of wounding as well as the potential role of wound factors in transformation.