RESUMO
Synaptic activity-dependent de novo gene transcription is crucial for long-lasting neuronal plasticity and long-term memory. In a forebrain neuronal conditional NF-kappaB-deficient mouse model, we demonstrate here that the transcription factor NF-kappaB regulates spatial memory formation, synaptic transmission, and plasticity. Gene profiling experiments and analysis of regulatory regions identified the alpha catalytic subunit of protein kinase A (PKA), an essential memory regulator, as a new NF-kappaB target gene. Consequently, NF-kappaB inhibition led to a decrease in forskolin-induced CREB phosphorylation. Collectively, these results disclose a novel hierarchical transcriptional network involving NF-kappaB, PKA, and CREB that leads to concerted nuclear transduction of synaptic signals in neurons, accounting for the critical function of NF-kappaB in learning and memory.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Memória/fisiologia , NF-kappa B/fisiologia , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ensaio de Desvio de Mobilidade Eletroforética , Eletrofisiologia , Perfilação da Expressão Gênica , Hipocampo/citologia , Hipocampo/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacosRESUMO
Nuclear factor kappaB (NF-kappaB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pylori pathogenesis, the role of NF-kappaB in murine cell responses to helicobacters has not been studied in detail. We thus investigated the abilities of different Helicobacter isolates to induce NF-kappaB-dependent responses in murine gastric epithelial cells (GECs) and in transgenic mice harboring an NF-kappaB-responsive lacZ reporter gene. H. pylori and Helicobacter felis strains up-regulated the synthesis in mouse GECs of the NF-kappaB-dependent chemokines KC (CXCL1) and MIP-2 (CXCL2). These responses were cag pathogenicity island (cagPAI) independent and could be abolished by pretreatment with a pharmacological inhibitor of NF-kappaB. Consistent with the in vitro data, experimental Helicobacter infection of transgenic mice resulted in increased numbers of GECs with nuclear beta-galactosidase activity, which is indicative of specific NF-kappaB activation. The numbers of beta-galactosidase-positive cells in mice were significantly increased at day 1 postinoculation with wild-type H. pylori strains harboring or not harboring a functional cagPAI, compared to naive animals (P = 0.007 and P = 0.04, respectively). Strikingly, however, no differences were observed in the levels of gastric NF-kappaB activation at day 1 postinoculation with H. felis or at day 30 or 135 postinoculation with H. pylori. This work demonstrates for the first time the induction of NF-kappaB activation within gastric mucosal cells during acute H. pylori infection. Furthermore, the data suggest that helicobacters may be able to regulate NF-kappaB signaling during chronic infection.
Assuntos
Células Epiteliais/microbiologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , NF-kappa B/metabolismo , Animais , Quimiocina CXCL1/biossíntese , Quimiocina CXCL2/biossíntese , Feminino , Mucosa Gástrica/imunologia , Mucosa Gástrica/microbiologia , Genes Reporter , Helicobacter felis/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/imunologia , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
The transcription factor Rel/nuclear factor (NF)-kappaB is known for its fundamental role in regulating immune and inflammatory responses. In the brain, constitutive NF-kappaB activity has been detected exclusively in neurons, and a large diversity of stimuli have been reported to induce NF-kappaB activity. Yet the function of this transcription factor in the nervous system remains unclear, and its role in neuroprotection or neurodegeneration is open to debate. Recently it was suggested that kappaB-driven gene expression in neurons is controlled by Sp1-like factors. To clarify such controversy, we have characterized here a novel mouse model in which the entire NF-kappaB-dependent transcriptional response is abolished in the forebrain. Calcium-calmodulin-dependent kinase II alpha promoter-driven tetracycline transactivator was used for regulated expression of a transdominant negative mutant of inhibitor kappaBalpha (super-repressor) together with a green fluorescent protein tracer. Inhibition of expression of a kappaB-dependent lacZ transgene was shown in triple transgenic mice, which correlated with the loss of kappaB-specific DNA binding. In transgenic organotypic hippocampal slice cultures, expression of the super-repressor led to strong cell death after neurotoxic insults. These data demonstrate for the first time that neuron-restricted ablation of NF-kappaB-driven gene expression increases neurodegeneration. This might lead to the path for new treatments of neurodegenerative diseases.
Assuntos
Regulação da Expressão Gênica , Proteínas I-kappa B/genética , NF-kappa B/antagonistas & inibidores , Neurônios/metabolismo , Prosencéfalo/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Morte Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Proteínas de Fluorescência Verde , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Proteínas I-kappa B/biossíntese , Técnicas In Vitro , Ácido Caínico/toxicidade , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Mutação , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Especificidade de Órgãos , Regiões Promotoras Genéticas , Prosencéfalo/citologia , TransgenesRESUMO
Mammary gland development starts in utero with one or several pairs of mammary rudiments (MRs) budding from the surface ectodermal component of the mammalian embryonic skin. Mice develop five pairs, numbered MR1 to MR5 from pectoral to inguinal position. We have previously shown that Gli3(Xt-J/Xt-J) mutant embryos, which lack the transcription factor Gli3, do not form MR3 and MR5. We show here that two days after the MRs emerge, Gli3(Xt-J/Xt-J) MR1 is 20% smaller, and Gli3(Xt-J/Xt-J) MR2 and MR4 are 50% smaller than their wild type (wt) counterparts. Moreover, while wt MRs sink into the underlying dermis, Gli3(Xt-J/Xt-J) MR4 and MR2 protrude outwardly, to different extents. To understand why each of these five pairs of functionally identical organs has its own, distinct response to the absence of Gli3, we determined which cellular mechanisms regulate growth of the individual MRs, and whether and how Gli3 regulates these mechanisms. We found a 5.5 to 10.7-fold lower cell proliferation rate in wt MRs compared to their adjacent surface ectoderm, indicating that MRs do not emerge or grow via locally enhanced cell proliferation. Cell-tracing experiments showed that surface ectodermal cells are recruited toward the positions where MRs emerge, and contribute to MR growth during at least two days. During the second day of MR development, peripheral cells within the MRs undergo hypertrophy, which also contributes to MR growth. Limited apoptotic cell death counterbalances MR growth. The relative contribution of each of these processes varies among the five MRs. Furthermore, each of these processes is impaired in the absence of Gli3, but to different extents in each MR. This differential involvement of Gli3 explains the variation in phenotype among Gli3(Xt-J/Xt-J) MRs, and may help to understand the variation in numbers and positions of mammary glands among mammals.
Assuntos
Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipertrofia , Fatores de Transcrição Kruppel-Like/fisiologia , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/fisiologia , Animais , Apoptose , Movimento Celular , Proliferação de Células , Camundongos , Proteína Gli3 com Dedos de ZincoRESUMO
Notch signaling is involved in numerous cell fate decisions in invertebrates and vertebrates. The Notch receptor is a type I transmembrane (TM) protein that undergoes two proteolytic steps after ligand binding, first by an ADAM (a distintegrin and metalloprotease) in the extracellular region, followed by gamma-secretase-mediated cleavage inside the TM domain. We demonstrate here that the murine ligand Delta1 (Dll1) undergoes the same sequence of cleavages, in an apparently signal-independent manner. Identification of the ADAM-mediated shedding site localized 10 aa N-terminal to the TM domain has enabled us to generate a noncleavable mutant. Kuzbanian/ADAM10 is involved in this processing event, but other proteases can probably substitute for it. We then show that Dll1 is part of a high-molecular-weight complex containing presenilin1 and undergoes further cleavage by a gamma-secretase-like activity, therefore releasing the intracellular domain that localizes in part to the nucleus. Using the shedding-resistant mutant, we demonstrate that this gamma-secretase cleavage depends on prior ectodomain shedding. Therefore Dll1 is a substrate for regulated intramembrane proteolysis, and its intracellular region possibly fulfills a specific function in the nucleus.
Assuntos
Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Vetores Genéticos , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Testes de Precipitina , Presenilina-1 , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , TransfecçãoRESUMO
The mouse develops five pairs of mammary glands that arise during mid-gestation from five pairs of placodes of ectodermal origin. We have investigated the molecular mechanisms of mammary placode development using Lef1 as a marker for the epithelial component of the placode, and mice deficient for Fgf10 or Fgfr2b, both of which fail to develop normal mammary glands. Mammary placode induction involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that FGF signaling is involved in the maintenance of mammary bud 4, and that Fgf10 deficient epithelium can undergo branching morphogenesis into the mammary fat pad precursor.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Glândulas Mamárias Animais/embriologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais , Animais , Feminino , Fator 10 de Crescimento de Fibroblastos , Deleção de Genes , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/fisiologia , Gravidez , Receptor Tipo 2 de Fator de Crescimento de FibroblastosRESUMO
Nuclear factor kappaB (NF-kappaB) transcription factors are key regulators of immune, inflammatory, and acute-phase responses and are also implicated in the control of cell proliferation and apoptosis. While perturbations in NF-kappaB activity impact strongly on B- and T-cell development, little is known about the role for NF-kappaB in natural killer (NK) cell differentiation. Inhibitors of NF-kappaB (IkappaBs) act to restrain NF-kappaB activation. We analyzed the cell-intrinsic effects of deficiencies in 2 IkappaB members (IkappaBalpha and IkappaBepsilon) on NK cell differentiation. Neither IkappaBalpha nor IkappaBepsilon deficiency had major effects on NK cell generation, while their combined absence led to NF-kappaB hyperactivation, resulting in reduced NK cell numbers, incomplete NK cell maturation, and defective interferon gamma (IFN-gamma) production. Complementary analysis of transgenic mice expressing an NF-kappaB-responsive reporter gene showed increased NF-kappaB activity at the stage of NK cell development corresponding to the partial block observed in IkappaBalpha x IkappaBepsilon-deficient mice. These results define a critical window in NK cell development in which NF-kappaB levels may be tightly controlled.
Assuntos
Proteínas I-kappa B/fisiologia , Células Matadoras Naturais/imunologia , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Quimera , Citocinas/biossíntese , Proteínas I-kappa B/genética , Células Matadoras Naturais/citologia , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Baço/imunologiaRESUMO
Delta1 acts as a membrane-bound ligand that interacts with the Notch receptor and plays a critical role in cell fate specification. By using peptide affinity chromatography followed by mass spectrometry, we have identified Dlg1 as a partner of the Delta1 C-terminal region. Dlg1 is a human homolog of the Drosophila Discs large tumor suppressor, a member of the membrane-associated guanylate kinase family of molecular scaffolds. We confirmed this interaction by co-immunoprecipitation experiments between endogenous Dlg1 and transduced Delta1 in a 3T3 cell line stably expressing Delta1. Moreover, we showed that deletion of a canonical C-terminal PDZ-binding motif (ATEV) in Delta1 abrogated this interaction. Delta4 also interacted with Dlg1, whereas Jagged1, another Notch ligand, did not. In HeLa cells, transfected Delta1 triggered the accumulation of endogenous Dlg1 at sites of cell-cell contact. Expression of Delta1 also reduced the motility of 3T3 cells. Finally, deletion of the ATEV motif totally abolished these effects but did not interfere with the ability of Delta1 to induce Notch signaling and T cell differentiation in co-culture experiments. These results point to a new, probably cell-autonomous function of Delta1, which is independent of its activity as a Notch ligand.