RESUMO
UDP-glucuronosyltransferases (UGTs) form a multigenic family of enzymes involved in the biotransformation and elimination of numerous endo- and xenobiotic compounds. Beside the diverse UGT isoforms present in the liver as well as in other tissues, the UGT2A1 isoform, also called olfactory UGT, was initially thought to be expressed in the nasal epithelium only. In this work, we demonstrate the UGT2A1 mRNA expression in the olfactory bulb, using in situ hybridization and quantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques. Within the epithelium, UGT2A1 mRNA is mainly found in the sustentacular cells and to a lesser extent in Bowman's gland cells. Moreover, in situ hybrization staining reveals UGT2A1 mRNA expression in the olfactory sensory neuron nuclei. Neuronal localization of UGT2A1 mRNA within the olfactory bulb is mainly found in the deeper granular cells. The development of the quantitative multistandard RT-PCR method firstly required characterization of the mouse Ugt2A1 cDNA by rapid amplification of cDNA ends (RACE)-PCR. UGT2A1 mRNA levels appear quantitatively six-fold lower in the olfactory bulb than in the epithelium, in both the rat and mouse. The expression of UGT2A1 in the olfactory bulb, which directly connects the nasal epithelium to the brain, emphasizes the potential role of this enzyme in the protection of the brain against airborne hazardous chemicals.
Assuntos
Glucuronosiltransferase/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Bulbo Olfatório/metabolismo , Mucosa Olfatória/metabolismo , Poluentes Atmosféricos/farmacocinética , Animais , Células Epiteliais/metabolismo , Glucuronosiltransferase/genética , Hibridização In Situ , Inativação Metabólica , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Neurônios Aferentes/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
The Ets family of transcription factors comprises several members which are involved to regulate gene transcription. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of effector cells to various target cell types. ICAM-1 is constitutively expressed at different levels in the absence of stimuli in different cell types, and its expression is upregulated by several proinflammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particularly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM significantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the first 176 bp upstream from the translational start site. Electrophoretic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions -158 and -138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM-1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Molécula 1 de Adesão Intercelular/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/genética , Coelhos , Transativadores/genética , Fatores de Transcrição/genética , Ativação Transcricional , Células Tumorais CultivadasRESUMO
The fusion of a wide range of proteins to the ligand-binding domain of nuclear receptors has been shown to impart ligand-dependent inducible activity of the resulting chimera. Transcriptional regulators of the ETS family are involved in both normal and oncogenic processes. In order to address the role of Erm, a "PEA3 subgroup" member of this family, we generated a chimera between Erm and the widely used ligand-binding domain of the oestrogen receptor (ER). The chimera, ErmER, consists of Erm protein fused at its C-terminal end to the ER domain. We show that ErmER displays a ligand-dependent transcriptional activity on ets responsive elements. The efficiency of ErmER mediated transactivation is modulated by the hormone concentration while its weak leakiness is reduced by using the steroidal anti-oestrogen EM-139. Our results define ErmER as the first conditional version of an Ets transcription factor, providing a useful tool to decipher Erm biological role and to identify potential Erm target genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Ligantes , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.
Assuntos
Proteínas de Ligação a DNA/genética , Família Multigênica , Proteínas Oncogênicas , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-ets , Sarcoma de Ewing/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
ERM, ER81 and PEA3 are three highly related transcription factors belonging to the ETS family. Together they form the PEA3 group within this family. Little data is yet available regarding the roles of these three genes during embryonic development. A prerequisite to investigations in this field is to obtain an accurate spatio-temporal expression map for the erm, er81 and pea3 genes. To this end, we have used in situ hybridization to compare their expression patterns during critical stages of murine embryogenesis. We report that all three genes are expressed in numerous developing organs coming from different embryonic tissues. The three genes appeared co-expressed in different organs but presented specific sites of expression, so that the resultant expression pattern could in fact reveal several distinct functions depending upon isolated and/or various combinations of the PEA3 member expression. These results suggest that erm, er81 and pea3 genes are differentially regulated, probably to serve important functions as cell proliferation control, tissue interaction mediator or cell differentiation, all over successive steps of the mouse organogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Sistema Nervoso Central/embriologia , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/genética , Ectoderma/metabolismo , Desenvolvimento Embrionário e Fetal , Endoderma/metabolismo , Expressão Gênica , Mesoderma/metabolismo , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição/genéticaRESUMO
H19 is an imprinted gene developmentally regulated in man and mouse and implicated in various neoplasms. No corresponding protein product has yet been detected, although several open reading frames (ORFs) could be identified along its RNA. The largest ORF found in the human gene could encode a putative 26 kDa protein. We have isolated two H19 cDNAs (AP and ES) that contain this ORF4 and correspond to incomplete copies of the unique 2.3 kb H19 RNA. In transient expression assays, AP was able to synthesize a 26 kDa protein whereas ES was not. With respect to ORF4, ES exhibits a 536 bp long GC-rich 5' untranslated region, whereas AP contains the last 22 nucleotides of this 5'UTR. Using deletions and point mutations, we have found that the length and probably the secondary structure of the 5'UTR strongly hampers the translatability of the RNA. In addition, a potential role of upstream ORFs (uORFs) was detected as stressed by the enhances translation of a construct mutated in uORF3 overlapping ORF4. Interactions between H19 and proteins are indicated by a specific binding between 5'UTR derived RNA segments and two nuclear proteins of about 27 kDa. Our results favor a potential role of these particular structures and binding properties in general trans-regulation of RNA post-transcriptional processes rather than in normal control of H19 mRNA translation.
Assuntos
Proteínas Musculares/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA não Traduzido , Sequências Reguladoras de Ácido Nucleico , Animais , Células COS , Galinhas , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Impressão Genômica , Humanos , Camundongos , Mutação , Proteínas Nucleares/classificação , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Filogenia , Ligação Proteica , RNA Longo não Codificante , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Ratos , Deleção de SequênciaRESUMO
17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17 beta-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17 beta-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal beta-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17 beta-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17 beta-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17 beta-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17 beta-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17 beta-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Enoil-CoA Hidratase , Isoenzimas/genética , Complexos Multienzimáticos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Expressão Gênica , Humanos , Hidroliases , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteína Multifuncional do Peroxissomo-2 , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Suínos , Distribuição TecidualRESUMO
Prognosis factors such as mutated or amplified oncogenes are used in the treatment of breast cancer. We have recently shown that the members of the PEA3 group (ER81, ERM and PEA3) from the transcription factor family of the ets genes are overexpressed in breast cancer tumors arising from MMTV-neu transgenic animals. Moreover, we have shown that ER81, and in a lesser extent ERM and PEA3, are not expressed in the estrogen and/or progesterone receptor-positive mammary cancer cell lines, whereas they are expressed in the receptor negative ones. Our research interest in now focused on the role(s) of these oncogenes in the development and the regulation of breast tumors.
Assuntos
Neoplasias da Mama/genética , Neoplasias Mamárias Experimentais/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Feminino , Humanos , Alinhamento de Sequência , Fatores de Transcrição/químicaRESUMO
We have investigated the activity of CMP-Neu5Ac:Gal beta 1-3GalNAc alpha-2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the beta-galactosidase alpha-2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activated ras gene decreases the activity of this specific alpha-2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of active O-glycan alpha-2,3-sialyltransferase polypeptides in ras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface of ras-transformed FR3T3 suggesting that no change in the sialylation of O-glycan core 1 appeared in these cells, although the activity of the alpha-2,3-sialyltransferase was decreased.