RESUMO
To investigate the function of NF-kappa B RelA (p65), we generated mice deficient in this NF-kappa B family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the SCID mice receiving relA-/- fetal liver transplants, similar to the relA+/+ and +/- cases. T cells were found to mature to Thy-1+/TCR alpha beta +/CD3+/CD4+ or CD8+, while B cells had the ability to differentiate to IgM+/B220+ and to secrete immunoglobulins. However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3 + anti-CD28, LPS, anti-IgM, and PMA + calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.
Assuntos
Imunoglobulina A/sangue , Imunoglobulina G/sangue , Ativação Linfocitária , Linfócitos/imunologia , NF-kappa B/deficiência , Animais , Linfócitos B/imunologia , Transplante de Células , Transplante de Tecido Fetal , Genes Letais , Heterozigoto , Homozigoto , Interleucina-2/biossíntese , Transplante de Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos SCID , NF-kappa B/genética , Receptores de Interleucina-2/biossíntese , Linfócitos T/imunologia , Fator de Transcrição RelARESUMO
Mice lacking the RelA (p65) subunit of NF-kappaB die between days 14 and 15 of embryogenesis because of massive liver destruction. Fibroblasts and macrophages isolated from relA-/- embryos were found to be highly sensitive to tumor necrosis factor (TNF) cytotoxicity, raising the possibility that endogenous TNF is the cause of liver cell apoptosis. To test this idea, we generated mice lacking both TNF and RelA. Embryogenesis proceeds normally in such mice, and TNF/RelA double-deficient mice are viable and have normal livers. Thus, the RelA-mediated antiapoptotic signal that protects normal cells from TNF injury in vitro can be shown to be operative in vivo.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , NF-kappa B/genética , Fator de Necrose Tumoral alfa/genética , Animais , Apoptose/genética , Desenvolvimento Embrionário e Fetal/imunologia , Morte Fetal/genética , Morte Fetal/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/imunologia , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The Delta/Serrate-Notch pathway is involved in intercellular signaling that controls cell fate during the development of invertebrates and vertebrates. Delta is a prototype of Notch ligands and has been studied extensively in Drosophila. In higher vertebrates, four Delta/Serrate homologues and four Notch homologues have been identified. Recent studies showed that the murine Delta homologue, mDelta1, is essential in early embryogenesis. The biological activity of mammalian Delta and its roles in cellular differentiation, however, have remained unclear. In this study, we first surveyed expression of mDelta1 in the adult mouse and found it to be present in a wide range of tissues. For testing biological activity of mDelta1, we expressed a mDelta1 full-length cDNA in L cells using a eukaryotic expression vector. Effects of mDelta1 on cellular differentiation were examined in two independent systems, featuring C2C12 myogenic differentiation and multipotent murine bone marrow cell differentiation. Inhibition of the former was observed with mDelta1 expression on L cells, associated with suppression of myogenin, a myogenic transcription factor. Expression of mDelta1 in conjunction with GM-CSF promoted differentiation of bone marrow cells to myeloid dendritic cells at the expense of other lineages. Although the effects of mDelta1 on two differentiation systems appeared opposing, as inhibition occurring in one and induction in the other, this can be understood by the unifying concept of generation of diverse cell types from equivalent progenitors. Thus, the present study provided evidence that mammalian Delta participates in intercellular signaling, determining the cell fate in a wide variety of tissues.
Assuntos
Diferenciação Celular , Proteínas de Membrana/fisiologia , Fatores de Transcrição , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Células Dendríticas/citologia , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Células L , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Proteínas Proto-Oncogênicas/biossíntese , Receptor Notch1 , Receptor Notch2 , Receptor Notch4 , Receptores de Superfície Celular/biossíntese , Receptores Notch , TransfecçãoRESUMO
p53 and NF-kappaB RelA are activated by various genotoxic agents and mutually suppress each other's ability to activate transcription, most likely through competition for transcriptional coactivators such as CBP or p300. However, we found that the inhibition by RelA of p53 transcriptional activity is not completely restored by CBP/p300 overexpression and that a p53 mutant can not suppress RelA activity despite of its ability to bind CBP/p300. In the present study, we further present evidence that these two transcriptional factors directly interact both in vivo and in vitro. These results therefore indicate that the cross transcriptional interference between p53 and RelA is partly caused by the direct interaction between these two transcription factors which is mediated by their dimerization/tetramerization domains and results in inhibition of each other's transcriptional activity. Finally, cells derived from RelA knockout mice showed enhanced p53 transcriptional activity, suggesting that this cross transcriptional interference is physiologically important in cellular response to genotoxic stress.