RESUMO
[This retracts the article DOI: 10.1128/JVI.56.1.284-292.1985.].
RESUMO
Three different Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are expressed in brain where they play a relevant role in maintaining Na+ and Ca2+ homeostasis. Although the neuroprotective roles of NCX2 and NCX3 in stroke have been elucidated, the relevance of NCX1 is still unknown because of embryonic lethality of its knocking-out, heart dysfunctions when it is overexpressed, and the lack of selectivity in currently available drugs. To overcome these limitations we generated two conditional genetically modified mice that upon tamoxifen administration showed a selective decrease or increase of NCX1 in cortical and hippocampal neurons. Interestingly, in cortex and hippocampus NCX1 overexpression increased, where NCX1 knock-out reduced, both exchanger activity and Akt1 phosphorylation, a neuronal survival signaling. More important, mice overexpressing NCX1 showed a reduced ischemic volume and an amelioration of focal and general deficits when subjected to transient middle cerebral artery occlusion. Conversely, NCX1-knock-out mice displayed a worsening of brain damage, focal and neurological deficits with a decrease in Akt phosphorylation. These results support the idea that NCX1 overexpression/activation may represent a feasible therapeutic opportunity in stroke intervention.
Assuntos
Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Córtex Cerebral/patologia , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Hipocampo/patologia , Masculino , Camundongos Knockout , Neurônios/patologia , Fosforilação , Trocador de Sódio e Cálcio/genética , Acidente Vascular Cerebral/genética , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Regulação para CimaRESUMO
The transcription factor Pax8 is already known to be essential at very early stages of mouse thyroid gland development, before the onset of thyroid hormone production. In this paper we show, using a conditional inactivation strategy, that the removal of the Pax8 protein late in gland development results in severe hypothyroidism, consequent to a reduced gland size and a deranged differentiation. These results demonstrate that Pax8 is also an essential player in controlling survival and differentiation of adult thyroid follicular cells.
Assuntos
Hipotireoidismo/genética , Fatores de Transcrição Box Pareados/genética , Glândula Tireoide/metabolismo , Tiroxina/genética , Animais , Diferenciação Celular , Sobrevivência Celular , Embrião de Mamíferos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hipotireoidismo/metabolismo , Hipotireoidismo/patologia , Hipotireoidismo/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/deficiência , Regiões Promotoras Genéticas , Transdução de Sinais , Glândula Tireoide/citologia , Glândula Tireoide/crescimento & desenvolvimento , Tiroxina/metabolismoRESUMO
BACKGROUND: Organogenesis of the thyroid gland requires the Pax8 protein. Absence or reduction of Pax8 results in congenital hypothyroidism in animal models and humans, respectively. This study aims at elucidating the regulatory mechanism leading to the expression of Pax8 in thyroid cells. METHODS: The murine Pax8 gene promoter was functionally dissected by mutagenesis and transfection in the thyroid cell line FRTL-5. Nuclear factors important for thyroid-specific gene expression were identified by DNA-binding assays. RESULTS: We show that Pax8 binds to and controls the expression of its own promoter. Furthermore, we identify a novel, thyroid-specific, DNA-binding activity (denominated nTTF [for novel Thyroid Transcription Factor]) that recognizes a specific region of the Pax8 promoter. CONCLUSIONS: The Pax8 promoter appears to be autoregulated, a feature that might be responsible for the haploinsufficiency displayed by this gene.
Assuntos
Homeostase/genética , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas , Glândula Tireoide/metabolismo , Animais , Sítios de Ligação/genética , Camundongos , Mutagênese , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/metabolismo , Ativação TranscricionalRESUMO
Regulated cell death, defined in morphological terms as apoptosis, is crucial for organ morphogenesis. While differentiation of the thyroid gland has been extensively studied, nothing is yet known about the survival mechanisms involved in the development of this endocrine gland. Using the zebrafish model system, we aim to understand whether genes belonging to the Bcl-2 family that control apoptosis are implicated in regulation of cell survival during thyroid development. Evidence of strong Bcl-2 gene expression in mouse thyroid precursors prompted us to investigate the functions played by its zebrafish homologs during thyroid development. We show that the bcl2-like (bcl2l) gene is expressed in the zebrafish thyroid primordium. Morpholino-mediated knockdown and mutant analyses revealed that bcl2l is crucial for thyroid cell survival and that this function is tightly modulated by the transcription factors pax2a, nk2.1a and hhex. Also, the bcl2l gene appears to control a caspase-3-dependent apoptotic mechanism during thyroid development. Thyroid precursor cells require an actively maintained survival mechanism to properly proceed through development. The bcl2l gene operates in the inhibition of cell death under direct regulation of a thyroid specific set of transcription factors. This is the first demonstration of an active mechanism to ensure survival of the thyroid primordium during morphogenesis.
Assuntos
Genes bcl-2 , Glândula Tireoide/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Proteína bcl-X/fisiologia , Animais , Sobrevivência Celular , Morfogênese , Glândula Tireoide/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Dicer is a type III ribonuclease required for the biogenesis of microRNAs (miRNAs), a class of small non-coding RNAs regulating gene expression at the post-transcriptional level. To explore the functional role of miRNAs in thyroid gland function, we generated a thyrocyte-specific Dicer conditional knockout mouse. Here we show that development and early differentiation of the thyroid gland are not affected by the absence of Dicer, while severe hypothyroidism gradually develops after birth, leading to reduced body weight and shortened life span. Histological and molecular characterization of knockout mice reveals a dramatic loss of the thyroid gland follicular architecture associated with functional aberrations and down-regulation of several differentiation markers. The data presented in this study show for the first time that an intact miRNAs processing machinery is essential for thyroid physiology, suggesting that deregulation of specific miRNAs could be also involved in human thyroid dysfunctions.
Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/metabolismo , Glândula Tireoide/enzimologia , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Ativação Enzimática , Imunofluorescência , Humanos , Hipotireoidismo/enzimologia , Hipotireoidismo/patologia , Camundongos , Camundongos Knockout , Morfogênese , Células Oxífilas/metabolismo , Células Oxífilas/patologia , Glândula Tireoide/crescimento & desenvolvimento , Glândula Tireoide/patologiaRESUMO
The thyroid and lungs originate as neighboring bud shaped outgrowths from the midline of the embryonic foregut. When and how organ specific programs regulate development into structures of distinct shapes, positions and functions is incompletely understood. To characterize, at least in part, the genetic basis of these events, we have employed laser capture microdissection and microarray analysis to define gene expression in the mouse thyroid and lung primordia at E10.5. By comparing the transcriptome of each bud to that of the whole embryo as well as to each other, we broadly describe the genes that are preferentially expressed in each developing organ as well as those with an enriched expression common to both. The results thus obtained provide a valuable resource for further analysis of genes previously unrecognized to participate in thyroid and lung morphogenesis and to discover organ specific as well as common developmental mechanisms. As an initial step in this direction we describe a regulatory pathway involving the anti-apoptotic gene Bcl2 that controls cell survival in early thyroid development.
Assuntos
Embrião de Mamíferos/metabolismo , Pulmão/metabolismo , Glândula Tireoide/metabolismo , Transcriptoma , Animais , Padronização Corporal/genética , Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Embrião de Mamíferos/embriologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Microdissecção e Captura a Laser , Pulmão/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Organogênese/genética , Glândula Tireoide/embriologia , Fatores de TempoRESUMO
In vertebrates the portion of the thyroid gland synthesizing the thyroid hormones develops from a small group of endodermal cells in the foregut. The nature of the signals that lead to the biochemical and morphogenetic events responsible for the organization of these cells into the adult thyroid gland has only recently become evident. In this review we summarize recent developments in the understanding of these processes, derived from evidence collected in several organisms.
Assuntos
Glândula Tireoide/embriologia , Animais , Sobrevivência Celular , Fatores de Crescimento de Fibroblastos/fisiologia , Humanos , Morfogênese , Proteínas Nucleares/fisiologia , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/fisiologia , Transdução de Sinais , Células-Tronco/citologia , Glândula Tireoide/citologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/fisiologia , Tretinoína/fisiologiaRESUMO
BACKGROUND: The transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation. RESULTS: Mouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1. CONCLUSIONS: The pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors.
Assuntos
Proteínas Nucleares/metabolismo , Hipófise/embriologia , Processamento de Proteína Pós-Traducional , Glândula Tireoide/embriologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Imunofluorescência , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genótipo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fosforilação , Estrutura Terciária de Proteína/genética , Deleção de Sequência , Relação Estrutura-Atividade , Glândula Tireoide/anormalidades , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene. RESULTS: We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it. CONCLUSIONS: Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control Pax8 transcription during thyroid differentiation and are relevant to further studies on Pax8 as a candidate gene for thyroid dysgenesis.
Assuntos
Elementos Facilitadores Genéticos/genética , Genômica , Fatores de Transcrição Box Pareados/genética , Glândula Tireoide/metabolismo , Região 5'-Flanqueadora/genética , Animais , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Especificidade de Órgãos , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/metabolismo , Filogenia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Glândula Tireoide/citologia , Fatores de Transcrição , Transcrição GênicaRESUMO
Activity of the sodium/iodide symporter (NIS) in lactating breast is essential for iodide (I(-)) accumulation in milk. Significant NIS upregulation was also reported in breast cancer, indicating a potential use of radioiodide treatment. All-trans-retinoic acid (tRA) is a potent ligand that enhances NIS expression in a subset of breast cancer cell lines and in experimental breast cancer models. Indirect tRA stimulation of NIS in breast cancer cells is very well documented; however, direct upregulation by tRA-activated nuclear receptors has not been identified yet. Aiming to uncover cis-acting elements directly regulating NIS expression, we screened evolutionary-conserved non-coding genomic sequences for responsiveness to tRA in MCF-7. Here, we report that a potent enhancer in the first intron of NIS mediates direct regulation by tRA-stimulated nuclear receptors. In vitro as well as in vivo DNA-protein interaction assays revealed direct association between retinoic acid receptor-alpha (RARalpha) and retinoid-X-receptor (RXR) with this enhancer. Moreover, using chromatin immunoprecipitation (ChIP) we uncovered early events of NIS transcription in response to tRA, which require the interaction of several novel intronic tRA responsive elements. These findings indicate a complex interplay between nuclear receptors, RNA Pol-II and multiple intronic RAREs in NIS gene, and they establish a novel mechanistic model for tRA-induced gene transcription.
Assuntos
Neoplasias da Mama/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Íntrons , Receptores do Ácido Retinoico/metabolismo , Simportadores/genética , Sequência de Bases , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Feminino , Genômica , Humanos , RNA Polimerase II/metabolismo , Elementos de Resposta , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides/metabolismo , Transcrição Gênica , Tretinoína/farmacologiaRESUMO
We report here the mapping of a chromosomal region responsible for strain-specific development of congenital hypothyroidism in mice heterozygous for null mutations in genes encoding Nkx2-1/Titf1 and Pax8. The two strains showing a differential predisposition to congenital hypothyroidism contain several single-nucleotide polymorphisms in this locus, one of which leads to a nonsynonymous amino acid change in a highly conserved region of Dnajc17, a member of the type III heat-shock protein-40 (Hsp40) family. We demonstrate that Dnajc17 is highly expressed in the thyroid bud and had an essential function in development, suggesting an important role of this protein in organogenesis and/or function of the thyroid gland.
Assuntos
Hipotireoidismo Congênito/genética , Predisposição Genética para Doença/genética , Proteínas de Choque Térmico HSP40/genética , Glândula Tireoide/anormalidades , Animais , Western Blotting , Células Cultivadas , Mapeamento Cromossômico , Cromossomos de Mamíferos/genética , Hipotireoidismo Congênito/metabolismo , Estudos de Associação Genética , Proteínas de Choque Térmico HSP40/metabolismo , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/metabolismo , Tireotropina/sangueRESUMO
A deranged differentiation is often a landmark of transformed cells. We used a thyroid cell line expressing an inducible Ras oncoprotein in order to study the hierarchy of molecular events leading to suppression of thyroid-specific gene expression. We find that, upon Ras activation, there is an immediate global down-regulation of thyroid differentiation, which is associated with an inhibition of the cAMP signaling pathway. We demonstrate that an unusual negative cross talk between Ras oncogene and the cAMP pathway induces inactivation of the transcription factor Pax8 that we propose as a crucial event in Ras-induced dedifferentiation.
Assuntos
Desdiferenciação Celular , AMP Cíclico/metabolismo , Genes ras , Fatores de Transcrição Box Pareados/metabolismo , Transdução de Sinais , Glândula Tireoide/citologia , Transcrição Gênica , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Elementos Facilitadores Genéticos/genética , Humanos , Cinética , Camundongos , Especificidade de Órgãos/genética , Fator de Transcrição PAX8 , Transporte Proteico , Ratos , Receptores da Tireotropina/metabolismo , Simportadores/metabolismoRESUMO
Kras is the most frequently mutated ras family member in lung carcinomas, whereas Hras mutations are common in tumors from stratified epithelia such as the skin. Using a Hras knock-in mouse model, we demonstrate that specificity for Kras mutations in lung and Hras mutations in skin tumors is determined by local regulatory elements in the target ras genes. Although the Kras 4A isoform is dispensable for mouse development, it is the most important isoform for lung carcinogenesis in vivo and for the inhibitory effect of wild-type (WT) Kras on the mutant allele. Kras 4A expression is detected in a subpopulation of normal lung epithelial cells, but at very low levels in lung tumors, suggesting that it may not be required for tumor progression. The two Kras isoforms undergo different post-translational modifications; therefore, these findings can have implications for the design of therapeutic strategies for inhibiting oncogenic Kras activity in human cancers.
Assuntos
Éxons/genética , Genes ras/fisiologia , Neoplasias Pulmonares/genética , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Sequências Reguladoras de Ácido Nucleico/genética , Neoplasias Cutâneas/genética , Animais , Carcinógenos/toxicidade , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/patologia , Uretana/toxicidadeRESUMO
Rhes is a small GTP-binding protein prominently localized in the striatum. Previous findings obtained in cell culture systems demonstrated an involvement of Rhes in cAMP/PKA signalling pathway, at a level proximal to the activation of heterotrimeric G-protein complex. However, its role in the striatum has been, so far, only supposed. Here we studied the involvement of Rhes in dopaminergic signalling, by employing mice with a null mutation in the Rhes gene. We demonstrated that the absence of Rhes modulates cAMP/PKA signalling in both striatopallidal and striatonigral projection neurons by increasing Golf protein levels and, in turn, influencing motor responses challenged by dopaminergic agonist/antagonist. Interestingly, we also show that Rhes is required for a correct dopamine-mediated GTP binding, a function mainly associated to stimulation of dopamine D2 receptors. Altogether, our results indicate that Rhes is an important modulator of dopaminergic transmission in the striatum.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Proteínas de Ligação ao GTP/genética , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Corpo Estriado/citologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espinhas Dendríticas/metabolismo , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Hipercinese/genética , Hipercinese/metabolismo , Hipercinese/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Neurônios/citologia , Técnicas de Cultura de Órgãos , Fenótipo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Sinapses/metabolismoRESUMO
In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility.
Assuntos
Ácido Aspártico/metabolismo , Cognição/fisiologia , Hipocampo/metabolismo , Potenciação de Longa Duração/genética , Animais , Ácido Aspártico/farmacologia , Cognição/efeitos dos fármacos , D-Aspartato Oxidase/genética , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Memória/fisiologia , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
Assuntos
Sequência de Bases/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Biblioteca Gênica , MicroRNAs/genética , Animais , Linhagem da Célula/genética , Sequência Conservada/genética , Neoplasias Hematológicas/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
In the thyroid, H(2)O(2) is produced at the apical pole of thyrocytes by one or two NADPH oxidases (NOX), Duox1/2 proteins. The onset of Duox expression was analysed by immunohistochemistry in the developing mouse thyroid in parallel with thyroglobulin (Tg) iodination and the expression of other thyroid differentiation markers. Duox proteins were found at embryonic day (E) 15.5 and were mainly localised at the apical pole of thyrocytes. Tg was detected 1 day before (E14.5) and Tg iodination was concomitant with the expression of both Duox and Na(+)/I(-) symporter (NIS; E15.5). The role of TSH in regulating Duox expression and H(2)O(2) accumulation was evaluated in thyroids of adult mice with reduced (Tshr(hyt/hyt) or mice treated with thyroxine) or increased (methimazole or perchlorate treatment) TSH/Tshr activity. In mice with suppressed TSH/Tshr activity, Duox expression was only partially decreased when compared with wild-type, as observed by western blot. In Tshr(hyt/hyt) strain, Duox was still expressed at the apical pole and H(2)O(2) measurements were normal. On the other hand, chronic TSH stimulation of the gland led to a decrease of H(2)O(2) measurements without affecting Duox expression. The onset of Duox protein expression is compatible with their proposed function in thyroid hormone synthesis and it can be considered as a functional marker of the developing thyroid. However, Duox expression in adult is much less regulated by TSH than NIS and thyroperoxidase. It is not always correlated with the overall thyroid H(2)O(2) accumulation, highlighting the importance of additional regulatory mechanisms which control either the production or H(2)O(2) degradation.
Assuntos
Flavoproteínas/análise , Regulação da Expressão Gênica no Desenvolvimento , Peróxido de Hidrogênio/análise , NADPH Oxidases/análise , Glândula Tireoide/embriologia , Animais , Western Blotting/métodos , Hipotireoidismo Congênito/embriologia , Hipotireoidismo Congênito/metabolismo , Oxidases Duais , Feminino , Imuno-Histoquímica , Iodeto Peroxidase/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Modelos Animais , Gravidez , Receptores da Tireotropina/metabolismo , Tireoglobulina/metabolismo , Glândula Tireoide/química , Glândula Tireoide/metabolismo , Tireotropina/sangue , Tireotropina/farmacologiaRESUMO
We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.