RESUMEN
T lymphocytes express two Src tyrosine kinases, Lck and Fyn. While thymocyte and T cell subsets are largely normal in fyn(-/-) mice, animals lacking Lck have impaired T cell development. Here, it is shown that Fyn is required for the rapid burst of interleukin (IL)-4 and IL-13 synthesis, which occurs promptly after T cell receptor activation. The lack of cytokine induction in fyn mutant mice is due to a block in natural killer (NK) T cell development. Studies using bone marrow chimeras indicate that the defect behaves in a cell-autonomous manner, and the lack of NK T cells is probably not caused by inappropriate microenvironmental cues. Both NK T cells and conventional T cells express similar levels of Lck, implying that Fyn and Lck have distinct roles in regulating NK T cell ontogeny. The fyn mutation defines the first signaling molecule that is selectively required for NK T cell, but not for T lymphocyte or NK cell development.
Asunto(s)
Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/enzimología , Familia-src Quinasas/genética , Animales , Complejo CD3/inmunología , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica/inmunología , Reordenamiento Génico/inmunología , Interleucina-13/biosíntesis , Interleucina-4/biosíntesis , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos , Mutación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn , Transducción de Señal/inmunología , Linfocitos T/enzimología , Linfocitos T/metabolismo , Familia-src Quinasas/inmunologíaRESUMEN
The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.
Asunto(s)
Adenosina Desaminasa/genética , Eritropoyesis , Células Madre Hematopoyéticas/citología , Hígado/embriología , Edición de ARN , Adenosina Desaminasa/metabolismo , Alelos , Animales , Quimera , Desarrollo Embrionario y Fetal , Eritroblastos/citología , Femenino , Células Madre Hematopoyéticas/enzimología , Hepatocitos/citología , Técnicas para Inmunoenzimas , Hígado/citología , Hígado/enzimología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Fenotipo , Proteínas de Unión al ARN , Células Madre/citología , Células Madre/enzimología , Teratoma/genética , Teratoma/patologíaRESUMEN
We analyzed the role of Fyn tyrosine kinase in CNS myelination by using fyn(-/-) null mutant mice, which express no Fyn protein. We found a severe myelin deficit in forebrain at all ages from 14 d to 1 year. The deficit was maximal at 1 month of age and was similar regardless of mouse strain background or whether it was determined by bulk isolation of myelin or by quantitation of myelin basic protein. To determine the cellular basis of the myelin deficit, we counted oligodendrocytes in tissue sections of mice expressing oligodendrocyte-targeted beta-galactosidase, and we used light and electron microscopy to examine the number and morphology of myelinated fibers and size of myelinated CNS structures. All of these parameters were reduced in fyn(-/-) mice. Unexpectedly, there were regional differences in the myelin deficit; in contrast to forebrain, fyn(-/-) cervical spinal cord exhibited no reduction in myelin content, number of oligodendrocytes, or number of myelinated fibers, nor was myelination delayed developmentally. We found that oligodendrocytes express Src, but there was no significant reduction of myelin content in null mutants lacking the Fyn-related kinases Src, Yes, or Lyn. Finally, we investigated the molecular features of Fyn that are required for myelination and found that a single amino acid substitution, which abolishes the tyrosine kinase activity of Fyn, resulted in a myelin deficit as great as that observed in the complete absence of Fyn protein. These results demonstrate that Fyn plays a unique role in myelination, one that requires its kinase activity.
Asunto(s)
Sistema Nervioso Central/metabolismo , Enfermedades Desmielinizantes/metabolismo , Vaina de Mielina/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Western Blotting , Recuento de Células , Células Cultivadas , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/patología , Cuerpo Calloso/crecimiento & desarrollo , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/patología , Genes Reporteros , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Prosencéfalo/crecimiento & desarrollo , Prosencéfalo/metabolismo , Prosencéfalo/patología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fyn , Proteínas Proto-Oncogénicas c-yes , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Médula Espinal/patología , Familia-src Quinasas/deficiencia , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Platelet basic protein (PBP) is a chemokine family member that is only found in platelets and their precursors megakaryocytes. The PBP gene is physically linked to the gene for another platelet-specific chemokine, platelet factor 4. While the biological basis of platelet factor 4 expression has been pursued by others, the regulatory features controlling the platelet-specific expression of PBP have not been investigated. In this article, we examined the molecular basis by which this megakaryocyte-specific gene is regulated. Transient expression studies of truncated reporter constructs containing from 4.5 to 0.1 kilobases of the functional PBP gene 5'-flanking region, demonstrated that the proximal 0.1 kilobases of the promoter was sufficient for high levels of expression in human erythroleukemia and CHRF-288 cells, two megakaryocytic cell lines. However, none of these constructs was expressed above background levels in HeLa and 293 cells, two non-megakaryocytic cell lines. Further truncation of this promoter suggested that there was an important regulatory element(s) within a pyrimidine-rich tract. Mobility shift analysis of the pyrimidine-rich tract defined a region between -85 and -64 which bound to a nuclear factor(s). This region contains sequences matching the consensus Ets-binding site from -78 to -75 base pairs. In particular, we noted that this site matched a PU.1 consensus sequence known as a PU box. Mobility shift and supershift studies with nuclear extracts as well as recombinant PU.1 protein and anti-PU.1 antibody further confirmed that PU.1 was the specific Ets family factor that bound to this site. Transient expression assays using reporter constructs which contained point mutations that abrogated PU.1 binding also significantly reduced PBP promoter activity in human erythroleukemia and CHRF cells. In addition, while all reporter gene constructs containing PBP promoters were completely inactive in HeLa cells, transactivation experiments using a PU.1 expression construct demonstrated that exogenous expression of PU.1 could increase reporter gene expression up to 8-fold in these cells. Finally, the role of PU.1 in PBP gene expression was compared between wild-type and PU.1-null embryonic stem (ES) cells that were differentiated in vitro into cells that resembled megakaryocytes both morphologically and immunologically. We found that PBP gene expression in the differentiated PU.1(-/-) null ES cells (as determined by semi-quantitative reverse transcriptase-polymerase chain reaction) was more than four times lower than that in the wild-type ES cells, while other platelet-specific genes were expressed equally or similarly in the two ES cell lines. Previous reports have shown that PU.1 is expressed in several hematopoietic lineages, including megakaryocytes. However, the functional role of PU.1 has only been previously demonstrated in the myeloid and lymphoid lineages. Therefore, our studies are the first to show the biological importance of this nuclear factor in the regulated expression of a megakaryocyte-specific gene.