RESUMO
We have identified a gene encoding a novel protein that is transcriptionally regulated by the Notch signaling pathway in mammals. This gene, named Nrarp (for Notch-regulated ankyrin-repeat protein), encodes a 114 amino acid protein that has a unique amino-terminus and a carboxy-terminal domain containing two ankyrin-repeat motifs. A Xenopus homolog of the Nrarp gene was previously identified in a large-scale in situ hybridization screen of randomly isolated cDNA clones. We demonstrate that in T-cell and myoblast cell lines expression of the Nrarp gene is induced by the intracellular domain of the Notch1 protein, and that this induction is mediated by a CBF1/Su(H)/Lag-1 (CSL)-dependent pathway. During mouse embryogenesis, the Nrarp gene is expressed in several tissues in which cellular differentiation is regulated by the Notch signaling pathway. Expression of the Nrarp gene is downregulated in Notch1 null mutant mouse embryos, indicating that expression of the Nrarp gene is regulated by the Notch pathway in vivo. Thus, Nrarp transcript levels are regulated by the level of Notch1 signaling in both cultured cell lines and mouse embryos. During somitogenesis, the Nrarp gene is expressed in a pattern that suggests that Nrarp expression may play a role in the formation of somites, and Nrarp expression in the paraxial mesoderm is altered in several Notch pathway mutants that exhibit defects in somite formation. These observations demonstrate that the Nrarp gene is an evolutionarily conserved transcriptional target of the Notch signaling pathway.
Assuntos
Anquirinas/química , Proteínas de Membrana/metabolismo , Proteínas/genética , Proteínas/fisiologia , Receptores de Superfície Celular , Transdução de Sinais , Fatores de Transcrição , Transcrição Gênica , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Diferenciação Celular , Linhagem Celular , Sistema Nervoso Central/embriologia , DNA Complementar/metabolismo , Regulação para Baixo , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Mesoderma/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Receptor Notch1 , Receptores Notch , Retroviridae/genética , Distribuição Tecidual , Células Tumorais Cultivadas , Xenopus , Proteínas de XenopusRESUMO
Notch proteins regulate many developmental processes. Notch1 is highly expressed on thymocytes, but its role in regulating their development is not known. We show that activation of Notch1 signaling in CD4+CD8+ double positive thymocytes promotes the maturation of both CD4+ and CD8+ single positive thymocytes and that this occurs in the absence of interactions between the T cell receptor and MHC molecules expressed on thymic epithelial cells. We have also identified several genes that are transcriptionally regulated by Notch1 in T cells and show that they are upregulated during maturation into both single positive lineages. These observations suggest that Notch1 signaling plays a role in promoting maturation into both the CD4 and CD8 T cell lineages.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proteínas de Transporte , Desintegrinas , Leucopoese , Proteínas de Membrana/metabolismo , Metaloendopeptidases , Metaloproteases , Receptores de Superfície Celular , Transdução de Sinais , Fatores de Transcrição , Proteínas ADAM , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Epiteliais , Proteínas de Homeodomínio/genética , Receptores de Hialuronatos/biossíntese , Hipoxantina Fosforribosiltransferase/genética , Complexo Principal de Histocompatibilidade , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Musculares/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas/genética , Receptor Notch1 , Receptores de Interleucina-2/biossíntese , Fatores de Transcrição HES-1 , Regulação para CimaRESUMO
Thymocyte development is a tightly regulated process. CD4+CD8+ double-positive (DP) immature thymocytes exhibit distinct phenotypic features from mature T cells; they express only 10% of surface TCR that are found on mature T cells and do not proliferate and produce IL-2 in response to stimulation. In this report we show that transgenic expression of the orphan nuclear receptor ROR gamma t in mature T cells down-regulates their surface TCR expression. The ROR gamma t transgene inhibits IL-2 production by mature T cells, and this inhibition may be partially due to the inhibitory effect of ROR gamma t on c-Rel transcription. Furthermore, ectopic expression of ROR gamma t inhibits the proliferation of mature and immature T cells. These results, together with its predominant expression in DP thymocytes, suggest that ROR gamma t controls these distinct phenotypic features of DP thymocytes. Our data suggest that down-regulation of ROR gamma t expression in thymocytes is essential for their maturation.
Assuntos
Regulação para Baixo/imunologia , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores do Ácido Retinoico , Receptores dos Hormônios Tireóideos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cruzamentos Genéticos , Regulação para Baixo/genética , Proteína Ligante Fas , Regulação da Expressão Gênica/imunologia , Imunossupressores/farmacologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Ligantes , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/biossíntese , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/metabolismo , Transgenes/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Receptor fas/metabolismoRESUMO
Notch signaling regulates cell fate decisions during development. Recent experiments suggest that Notch signaling is essential for initial commitment to the T cell lineage and may function together with signals from the pre-TCR and the TCR to regulate subsequent steps of T cell development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Células-Tronco Hematopoéticas/citologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Subpopulações de Linfócitos T/citologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Linhagem da Célula , Retroalimentação , Hematopoese/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Receptor Notch1 , Receptor Notch2 , Receptor Notch4 , Receptores de Antígenos de Linfócitos T/genética , Receptores de Superfície Celular/genética , Receptores Notch , Transdução de Sinais , Timo/citologia , Fatores de Transcrição/classificação , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
The Notch receptor and its ligands are involved in many developmental processes. They are highly expressed in the thymus and have been implicated in the CD4 versus CD8 lineage decision. We identified the constitutively active intracellular fragment of murine Notch-1 as capable of rendering thymomas resistant to glucocorticoid-induced apoptosis. This effect was confirmed in other T cell lines and in CD4+ CD8+ DP thymocytes. Activation of the Notch signaling pathway also upregulated a number of other markers that, like steroid resistance, correlate with DP maturation into both the CD4 and CD8 lineages. These results suggest that Notch signaling is critically involved in the maturation of DP thymocytes into both CD4+ and CD8+ SP thymocytes.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Drosophila , Leucopoese , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Fatores de Transcrição , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Resistência a Medicamentos , Glucocorticoides/farmacologia , Proteínas de Insetos/genética , Líquido Intracelular , Camundongos , Mapeamento de Peptídeos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptor Notch1 , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Timo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
We have identified RORgamma t, a novel, thymus-specific isoform of the orphan nuclear receptor RORgamma that is expressed predominantly in CD4+ CD8+ double-positive thymocytes. Ectopic expression of RORgamma t protects T cell hybridomas from activation-induced cell death by inhibiting the upregulation of Fas ligand. Following hybridoma stimulation, RORgamma t also inhibits IL-2 production but does not affect the induction of Nur-77 and Egr-3 nor the upregulation of CD69. Both the ligand-binding and DNA-binding domains of RORgamma t are required for this effect. We propose that the role of RORgamma t expression in immature thymocytes is to inhibit Fas ligand expression and cytokine secretion following engagement of their TCR during positive or negative selection.