RESUMO
The products of the collagen-alpha 1(I) and -alpha 2(I) genes form the triple helical molecule collagen type I, which constitutes the major ECM protein in tissue fibrosis. The collagen-alpha 1(I) gene is mainly transcriptionally regulated, and its promoter activity depends on the interaction of the transcription factors NF-I and Sp1 with a tandem repeat of evolutionary conserved NF-I/Sp1 switch elements. An increased affinity of Sp1 to these elements has been observed in experimental liver fibrosis. Here, we demonstrate that the DNA binding drug mithramycin displays a high affinity binding to the GC-rich elements in the collagen-alpha 1(I) promoter as measured by DNAse I protection and gel retardation assays. Mithramycin interferes with Sp1 but not with NF-I binding to these sites. At a concentration of 100 nM, mithramycin efficiently reduces basal and TGF-beta-stimulated alpha 1(I) gene expression in human primary fibroblasts. The transcriptional activity and mRNA steady state levels of other genes, including the collagenase gene, as well as the growth rate of fibroblasts remained unchanged on exposure to this drug. Taken together, our results indicate that the transcriptional activity of the type I collagen gene highly depends on its GC-rich regulatory elements, and further, that these elements can be differentially blocked, thereby changing the balance between ECM structural and degrading gene activities in human fibroblasts.
Assuntos
Colágeno/biossíntese , Expressão Gênica/efeitos dos fármacos , Plicamicina/toxicidade , Northern Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Sondas de DNA , DNA Complementar/análise , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genes jun , Humanos , Cinética , Pulmão/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , RNA/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Colágeno/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/fisiologia , Desoxirribonuclease I , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Transcrição NFI , Proteínas Nucleares/metabolismo , Sondas de Oligonucleotídeos , Plasmídeos , Ligação Proteica , Transfecção , Proteína 1 de Ligação a Y-BoxRESUMO
The nemo-like kinase (Nlk) connects the MAP kinase and Wnt signalling pathways. We have found that invertebrate (Caenorhabditis elegans, Drosophila melanogaster) and mammalian genomes (Mus musculus and Homo sapiens) each contain only a single functional Nlk gene. The mouse genome also harbours a transcriptionally silent processed Nlk pseudogene residing on chromosome 2. Thus, while genes encoding upstream (such as Wnts and frizzelds) and downstream (such as TCF/LEF) components of the Wnt signalling pathway have been extensively diversified during evolution, genes encoding components of the common core of the connecting signalling structure (such as beta-catenin, GSK beta and Nlk) have been maintained in single copies.
Assuntos
Genoma , Proteínas Quinases Ativadas por Mitógeno/genética , Sequência de Aminoácidos , Animais , Southern Blotting , Caenorhabditis elegans/genética , DNA/genética , Drosophila melanogaster/genética , Humanos , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases , Pseudogenes/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The expression of type I collagen is regulated developmentally and tissue specifically. Two sets of binding sites for nuclear factor I (NF-I) and Sp1 transcription factors arrayed as an imperfect tandem repeat are critical for high activity of the murine alpha 1(I) collagen gene in NIH-3T3 fibroblasts and are conserved in evolution. Gel retardation analysis combined with methylation interference studies show that NF-I and Sp1 bind to overlapping sites in a mutually exclusive manner. Cotransfection studies using Drosophila Schneider L2 cells, which lack both transcription factors, demonstrate that each factor alone trans-activates the gene, while cotransfection of both factors results in the inhibition of the strong Sp1 trans-activation. In contrast, the herpes simplex virus thymidine kinase promoter, which contains functionally independent NF-I and Sp1 binding sites, is maximally transactivated by the cotransfection of both factors. Because the two NF-I/Sp1 binding sites overlap, the ratio of the activities of the two factors rather than their absolute concentrations determine alpha 1(I) gene expression, characterizing these promoter sequences as transcription factor switch elements.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Colágeno/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação , DNA , Drosophila/genética , Metilação , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFI , Proteínas Nucleares , Regiões Promotoras Genéticas , Ativação Transcricional , Transfecção , Proteína 1 de Ligação a Y-BoxRESUMO
One component of host defense at mucosal surfaces is epithelium-derived peptides with antimicrobial activity called defensins. We describe in this report the isolation and characterization of a murine homologue of human beta-defensin 2 (hBD-2) called mouse beta-defensin 3 (mBD-3). The predicted amino acid sequence shows the hallmark features of other known epithelial defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-3 revealed two exons separated by a 1.7-kb intron. The mBD-3 gene is localized at the proximal portion of chromosome 8, the site where genes for mouse alpha- and beta-defensins are found. Under basal condition, mBD-3 transcripts were detected at low levels in epithelial cells of surface organs, such as the intestine and lung. After instillation of Pseudomonas aeruginosa PAO1 into mouse airways, mBD-3-specific mRNA was upregulated significantly not only in large airways but also in the small bowel and liver. Recombinant mBD-3 peptide, produced from a baculovirus expression system, showed antimicrobial activity against P. aeruginosa PAO1 (MIC of 8 micrograms/ml) and Escherichia coli D31 (MIC of 16 micrograms/ml) in a salt-dependent manner. This study demonstrates that a murine homologue of hBD-2 is present in the respiratory system and other mucosal surfaces. These similarities between murine and human host defense apparatus provide further impetus to evaluate the mouse as a model for studying the human innate host defense system.
Assuntos
Células Epiteliais/imunologia , Infecções por Escherichia coli/imunologia , Biossíntese de Proteínas , Proteínas/imunologia , Infecções por Pseudomonas/imunologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/imunologia , Sequência de Bases , Defensinas , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas/genética , Alinhamento de SequênciaRESUMO
Cystinuria, one of the most common inborn errors of metabolism in humans, accounts for 1-2% of all cases of renal lithiasis. It is caused by defects in the heterodimeric transporter system rBAT/b0,+AT, which lead to reduced reabsorption of cystine and dibasic amino acids through the epithelial cells of the renal tubules and the intestine. In an N-ethyl-N-nitrosourea mutagenesis screen for recessive mutations we identified a mutant mouse with elevated concentrations of lysine, arginine and ornithine in urine, displaying the clinical syndrome of urolithiasis and its complications. Positional cloning of the causative mutation identified a missense mutation in the solute carrier family 3 member 1 gene (Slc3a1) leading to an amino acid exchange D140G in the extracellular domain of the rBAT protein. The mouse model mimics the aetiology and clinical manifestations of human cystinuria type I, and is suitable for the study of its pathophysiology as well as the evaluation of therapeutic and metaphylactic approaches.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos , Proteínas de Transporte/fisiologia , Cistina/metabolismo , Cistinúria/etiologia , Modelos Animais de Doenças , Glicoproteínas de Membrana/fisiologia , Cálculos da Bexiga Urinária/patologia , Cálculos Urinários/etiologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Arginina/urina , Proteínas de Transporte/genética , Mapeamento Cromossômico , Cistinúria/genética , Cistinúria/patologia , Etilnitrosoureia , Feminino , Genótipo , Lisina/urina , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese , Mutação , Ornitina/urina , Fenótipo , Homologia de Sequência de Aminoácidos , Cálculos Urinários/genética , Cálculos Urinários/patologiaRESUMO
The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.