RESUMEN
RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL(-/-) mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2(S5A)Tg mice, Id2(S5A) (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2(S5A) rescued increased apoptosis and defective differentiation of MECs in RANKL(-/-) mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.
Asunto(s)
Diferenciación Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Células Epiteliales/fisiología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Animales , Línea Celular Tumoral , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Lactancia , Masculino , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Proteínas de la Leche/genética , Proteínas de la Leche/metabolismo , Fosforilación , Embarazo , Transporte de Proteínas , Receptor Activador del Factor Nuclear kappa-B/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: The intestinal epithelium consists of EphB2-positive proliferative basal cryptic cells and EphrinB1-positive, postmitotic differentiated cells. We investigated the effects of Notch signaling on formation of the EphB2-EphrinB1 boundary using mouse and tissue culture models. METHODS: We created mice in which Mind bomb-1 (Mib1), an essential E3 ubiquitin ligase that activates Notch ligands, was inactivated specifically in the intestinal epithelia (Vil-Cre;Mib1(f/f)); Notch is, therefore, inactivated in this tissue. We also studied the effects of different inhibitors on intestinal epithelial cells (IEC-6) that express activated Notch. Tissues and cells were analyzed by immunohistochemical and immunoblot analyses. RESULTS: The intestinal epithelia of Vil-Cre;Mib1(f/f) mice had reduced numbers of EphrinB1-positive cells, compared with controls, but increases in EphB2-positive cells; beta-catenin was activated in these cells. These phenotypes were reversed by expression of a constitutively active form of Notch1. In the IEC-6 cells, Notch signaling activated the expression of EphrinB1 in an Hes1-independent manner, but down-regulated the expression of EphB2 through the GSK3beta-mediated inhibition of beta-catenin. CONCLUSIONS: Notch signaling regulates formation of the EphB2-EphrinB1 boundary in the mouse intestinal epithelium.
Asunto(s)
Efrina-B1/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Células Madre/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proliferación Celular , Efrina-B2/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fosforilación , Receptor Notch1/genética , Factor de Transcripción HES-1 , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is a transcriptional factor that performs a broad spectrum of biological functions in response to various stimuli. However, no specific coactivator that regulates the transcriptional activity of STAT3 has been identified. Here we report that CR6-interacting factor 1 (Crif1) is a specific transcriptional coactivator of STAT3, but not of STAT1 or STAT5a. Crif1 interacts with STAT3 and positively regulates its transcriptional activity. Crif1-/- embryos were lethal around embryonic day 6.5, and manifested developmental arrest accompanied with defective proliferation and massive apoptosis. The expression of STAT3 target genes was markedly reduced in a Crif1-/- blastocyst culture and in Oncostatin M-stimulated Crif1-deficient MEFs. Importantly, the key activities of constitutively active STAT3-C, such as transcription, DNA binding, and cellular transformation, were abolished in the Crif1-null MEFs, suggesting the essential role of Crif1 in the transcriptional activity of STAT3. Our results reveal that Crif1 is a novel and essential transcriptional coactivator of STAT3 that modulates its DNA binding ability, and shed light on the regulation of oncogenic STAT3.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Apoptosis , Blastocisto/metabolismo , Blastocisto/patología , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , ADN/metabolismo , Femenino , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Embarazo , Factor de Transcripción STAT3/genética , Transcripción GenéticaRESUMEN
The Delta-Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for cell fate specification. Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. We now report that mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulated in the plasma membrane, while it was localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. Our results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling.