Asunto(s)

Infecciones por Coronavirus , Genoma , Pandemias , Neumonía Viral , Betacoronavirus , COVID-19 , Humanos , Filogenia , SARS-CoV-2

RESUMEN

Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.

Asunto(s)

Tipificación del Cuerpo , Ayuno/fisiología , Conducta Alimentaria/fisiología , Proteínas del Helminto/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Turbelarios/embriología , Turbelarios/fisiología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Clonación Molecular , Regulación de la Expresión Génica , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Filogenia , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Alineación de Secuencia , Turbelarios/genética

Asunto(s)

Informática , Humanos , Hidroximetilbilano Sintasa/genética , Internet , Mutación/genética , Porfiria Intermitente Aguda/enzimología , Porfiria Intermitente Aguda/terapia

RESUMEN

The C. elegans genome encodes an unexpectedly large number of NHRs, the majority of which are classified as supplementary nuclear receptors (supnrs) that are likely to have evolved from an ancestral protein related to vertebrate HNF-4. To understand the need for this large repertoire of potential ligand-activated transcription factors, we have begun to study an 18-member subgroup defined by DNA binding domain relatedness. Here we report on NHR-60, a supnr expressed ubiquitously throughout development with a distinct pattern of localization on the nuclear periphery. Both antibody staining and GFP reporter genes demonstrated high-level expression and accumulation of NHR-60 in seam cell nuclei that is dependent on NHR-23 activity. Interference with NHR-60 activity, by either RNAi or overexpression of a putative dominant negative isoform, results in embryonic and early larval lethality, including defects in seam cell development. This adds NHR-60 to the list of C. elegans NHRs playing important roles in development.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Especificidad de Anticuerpos , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Embrión no Mamífero/citología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Genes Dominantes , Genes Reporteros , Genoma de los Helmintos/genética , Proteínas Fluorescentes Verdes/metabolismo , Larva/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Valproic acid (2-propyl pentanoic acid) is a drug used for the treatment of epilepsy and bipolar disorder. Although very rare, side effects such as spina bifida and other defects of neural tube closure indicate that valproic acid interferes with developmental regulatory pathways. Recently obtained data show that valproic acid affects cell growth, differentiation, apoptosis and immunogenicity of cultured cancer cells and tumours. Focused studies uncovered the potential of valproic acid to interfere with multiple regulatory mechanisms including histone deacetylases, GSK3 alpha and beta, Akt, the ERK pathway, the phosphoinositol pathway, the tricarboxylic acid cycle, GABA, and the OXPHOS system. Valproic acid is emerging as a potential anticancer drug and may also serve as a molecular lead that can help design drugs with more specific and more potent effects on the one side and drugs with wide additive but weaker effects on the other. Valproic acid is thus a powerful molecular tool for better understanding and therapeutic targeting of pathways that regulate the behaviour of cancer cells.

Asunto(s)

Antineoplásicos/farmacología , Ácido Valproico/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Teratógenos/toxicidad , Ácido Valproico/análogos & derivados , Ácido Valproico/química , Ácido Valproico/uso terapéutico

RESUMEN

BIR-1 and Survivin are highly conserved members of the inhibitor of apoptosis protein family that regulate cell division in nematodes and mammals and inhibit apoptosis in mammals. In the C. elegans genome, bir-1 is organized in an operon together with transcription and splicing cofactor CeSKIP (skp-1) and is highly expressed during embryogenesis as well as in non-dividing cells during larval development. Previously we have shown that BIR-1 regulates transcription and development and its loss-of-function phenotype overlaps with loss of function of CeSKIP and nuclear hormone receptor CHR3 (NHR-23). Here we searched for genes whose expression is affected by BIR-1 loss of function using whole-genome microarray experiments and identified several collagen genes as candidate targets of bir-1 inhibition in L1 larval stage. The decreased expression of selected collagen genes in bir-l-inhibited larvae was confirmed by quantitative RT-PCR. Next, we generated transgenic lines expressing bir-1 mRNA under a heat shock-regulated promoter and tested whether bir-1 overexpression has the potential to augment the expression of genes that showed decreased expression in worms treated with bir-1 RNAi. Overexpression of bir-1 resulted in a pronounced increase (2 to 5 times) of the expression of these genes. Our findings support the concept that BIR-1, a protein generally regarded as a mitotic factor, is involved in the regulation of transcription during normal development of C. elegans and has a strong ability to affect transcription of developmentally active genes if overexpressed.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Colágeno/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Homología de Secuencia , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Perfilación de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , Larva/metabolismo , Análisis por Micromatrices , Interferencia de ARN , Reproducibilidad de los Resultados , Survivin , Transcripción Genética

RESUMEN

Abnormal expression of histone deacetylases may contribute to the establishment of a cancer specific transcription profile. We examined expression of HDAC3 in human non-malignant gliosis and glial astrocytic tumours. Samples from four non-malignant gliosis and 17 astrocytic gliomas (six of grade II, one of grade III and ten of grade IV) removed for therapeutic purposes were assayed for HDAC3 expression at mRNA and protein levels. HDAC3 mRNA was detected in non-tumorous gliosis as well as in all examined glial tumours. Seven out of eleven examined high-grade tumours showed an elevated number of copies of HDAC3 mRNA. Western blot analysis detected high levels of expression of HDAC3 in the majority of the examined tumours. Immunohistochemistry and immunofluorescence made on a collection of 35 astrocytic tumours detected nuclear as well as cytoplasmic HDAC3 expression in all of those tumours. While the distribution of HDAC3 was both nuclear as well as cytoplasmic and moderate in intensity in non-malignant tissues and low-grade gliomas, high-grade tumours expressed HDAC3 in a focally deregulated pattern that included strongly pronounced cytoplasmic localization. Confocal microscopy and additional co-localization analysis detected nuclear HDAC3 in all tumours examined. We conclude that HDAC3 expression is elevated in human astrocytic tumours and its expression pattern is deregulated at the cellular level in high-grade gliomas.

Asunto(s)

Astrocitoma/enzimología , Neoplasias Encefálicas/enzimología , Histona Desacetilasas/metabolismo , Secuencia de Aminoácidos , Astrocitoma/genética , Neoplasias Encefálicas/genética , Núcleo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Gliosis/metabolismo , Histona Desacetilasas/genética , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo

RESUMEN

CD44 comprises a family of membrane adhesion molecules encoded by a single gene and diversified by alternative splicing and extensive posttranslational modifications. Alterations of CD44 expression patterns are linked to tumour invasion and formation of metastases. However, CD44 expression and its relation to the biological properties of tumours vary depending on the tumour type and origin. In transitional cell carcinoma of the urinary bladder, low CD44 expression is linked to enhanced tumour aggressiveness. We studied CD44 expression in two urothelial cancer cell lines, HT1197 and 5637. CD44s and a v6 variable exon-containing splice variants were detected in both cell lines by reverse transcription-PCR and by commercially available monoclonal antibodies. In both cell lines, Western blot analysis detected immunoreactive proteins with approximate sizes 70-85 kD, 95-110 kD, and 120-140 kD with CD44v6 antibody and weak bands with size 70-98 kD with CD44s antibody. At the cellular level, the pattern of CD44 immunoreactivity correlated with a lower level of cell differentiation and a higher degree of cell proliferation. In HT1197 cells, the CD44v6 was detected predominantly in small proliferating cells and in large multinuclear atypical cells. CD44s and CD44v6 displayed low immunoreactivity in HT1197 cells with a higher degree of epithelial differentiation. The 5637 cells expressed CD44v6 strongly and CD44s weakly. We conclude that CD44v6 expression correlates with a higher proliferative activity and with a stem cell-like phenotype in both cell lines and with cellular atypia in HT1197 cells.

Asunto(s)

Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/patología , Glicoproteínas/metabolismo , Receptores de Hialuranos/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Urotelio/metabolismo , Urotelio/patología , Empalme Alternativo/genética , Empalme Alternativo/inmunología , Carcinoma de Células Transicionales/fisiopatología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Proliferación Celular , Forma de la Célula/genética , Forma de la Célula/inmunología , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Inmunohistoquímica , Invasividad Neoplásica/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Neoplasias de la Vejiga Urinaria/fisiopatología

RESUMEN

CHR3 (nhr-23, NF1F4), the homologue of Drosophila DHR3 and mammalian ROR/RZR/RevErbA nuclear hormone receptors, is important for proper epidermal development and molting in the nematode Caenorhabditis elegans. Disruption of CHR3 (nhr-23) function leads to developmental changes, including incomplete molting and a short, fat (dumpy) phenotype. Here, we studied the role of CHR3 during larval development by using expression assays and RNA-mediated interference. We show that the levels of expression of CHR3 (nhr-23) cycle during larval development and reduction of CHR3 function during each intermolt period result in defects at all subsequent molts. Assaying candidate gene expression in populations of animals treated with CHR3 (nhr-23) RNA-mediated interference has identified dpy-7 as a potential gene acting downstream of CHR3. These results define CHR3 as a critical regulator of all C. elegans molts and begin to define the molecular pathway for its function.

Asunto(s)

Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos , Muda , Receptores de Péptidos de Invertebrados/fisiología , Transactivadores , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Larva , Receptores de Péptidos de Invertebrados/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temperatura

RESUMEN

Because they are sparsely distributed in tissues, dendritic cells (DC) present in nonlymphoid organs are difficult to isolate. Only DC from skin and lung have been successfully studied in culture. The objective of the present work was to investigate the possibility of isolating and culturing DC from an endocrine organ, the thyroid gland, which is particularly susceptible to the development of autoimmune processes. The study was conducted on pig thyroid glands to have sufficient amounts of starting material. This choice required the characterization of immunological reagents capable of recognizing DC markers in the pig species. Using a discontinuous trypsinization procedure, a DC population representing 2% to 3% of the thyroid cell suspension was reproducibly obtained. Isolated DC quantitatively attached to tissue culture-treated dishes and segregated from thyrocytes. DC identified as cells expressing major histocompatibility complex class II molecules, the mannose receptor, and the S100 protein were found to have a high capacity to internalize labeled ligands, dextran, and mannosylated albumin. These cells had a phenotype of immature DC. Secondarily, a fraction of DC detached from culture dishes, and floating DC had low or no endocytic activity, a characteristic of mature DC. Treatment of DC/thyrocytes cocultures with tumor necrosis factor alpha (TNFalpha) activated the transformation of immature DC into mature DC. These data show that DC isolated from the thyroid gland can be maintained immature or activated to undergo maturation in primary culture. The procedure of cell isolation and culture should be adaptable to human thyroid tissue for in vitro analyses of DC-mediated immune responses.

Asunto(s)

Células Dendríticas/citología , Glándula Tiroides/citología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Células Dendríticas/ultraestructura , Humanos , Hidrólisis , Microscopía Electrónica , Fenotipo , Porcinos , Glándula Tiroides/ultraestructura

RESUMEN

Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian, Tripedalia cystophora, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-cis retinoic acid (Kd = 4 x 10(-10) M) and binds to the DNA sequence, PuGGTCA as a monomer in vitro. jRXR also heterodimerizes with Xenopus TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His6)jRXR fusion protein was identified in the promoters of three T. cystophora crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate in vivo targets for jRXR. Finally, an antibody prepared against (His6)jRXR showed that full-length jRXR is expressed at all developmental stages of T. cystophora except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.

Asunto(s)

Cnidarios/metabolismo , ADN Complementario/genética , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cnidarios/genética , ADN Complementario/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes de Fusión/genética

RESUMEN

CHR3 is a Caenorhabditis elegans orphan nuclear hormone receptor highly homologous to Drosophila DHR3, an ecdysone-inducible gene product involved in metamorphosis. Related vertebrate factors include RORalpha/RZRalpha, RZRbeta and RevErb. Gel-shift studies show that CHR3 can bind the DR5-type hormone response sequence. CHR3 is a nuclear protein present in all blastomeres during early embryogenesis. During morphogenesis, both CHR3 protein and zygotically active reporter genes are detectable in epidermal cells and their precursors. Inhibition of the gene encoding CHR3 results in several larval defects associated with abnormal epidermal cell function, including molting and body size regulation, suggesting that CHR3 is an essential epidermal factor required for proper postembryonic development.

Asunto(s)

Caenorhabditis elegans/embriología , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/genética , ADN de Helmintos/metabolismo , Epidermis/química , Epidermis/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes de Helminto/fisiología , Proteínas de Choque Térmico/genética , Larva , Muda , Regiones Promotoras Genéticas/genética , ARN sin Sentido , ARN de Helminto/análisis , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión

RESUMEN

Orphan nuclear receptors such as Nurr1 and Nur77 have conserved amino acid sequences in the zinc finger DNA binding domains and similar COOH-terminal regions, but have no known ligands. These receptors can bind DNA sequences (response elements) as monomers and can also heterodimerize with the retinoid X receptor to activate transcription. We report here the identification and initial characterization of a novel COOH-terminal truncated isoform of Nurr1, Nurr1a. Internal splicing of Nurr1 generates a frameshift such that a stop codon is prematurely encoded resulting in a naturally occurring COOH-terminal truncation. Embryonic and postnatal mouse brain showed both Nurr1 and Nurr1a mRNAs expressed during development. To characterize essential COOH-terminal elements that may be deleted from Nurr1a and determine function in putative ligand binding, we created COOH-terminal deletion mutants. Nurr1, Nur77, and 3'-truncated mutants bind in gel mobility shift assays to the monomeric Nur77 response element (B1A-RE). However, in transient transfection assays, a truncation of as little as 15 Nurr1 COOH-terminal amino acids diminished transcriptional activation of B1A-thymidine kinase-chloramphenicol acetyltransferase reporter. This result was not seen for a similar Nur77 deletion mutant, Nur77-586. Unlike full-length Nurr1 and Nur77, transactivation by Nur77-586 was not augmented in response to the presence of retinoid-like receptor and 9-cis-retinoic acid. Thus, the interaction of putative ligand binding and transactivation for Nurr1 and Nur77 may function differently.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Células 3T3 , Alitretinoína , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Clonación Molecular , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores Citoplasmáticos y Nucleares , Receptores de Ácido Retinoico/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Receptores X Retinoide , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/genética , Tretinoina/metabolismo

RESUMEN

Serum proteins such as albumin are present inside thyroid follicles in both normal and pathological situations. To analyze the mechanism of entry of these proteins, we investigated the ability of polarized thyrocytes to internalize soluble molecules at their basolateral pole. Experiments were conducted on in vitro reconstituted thyroid follicles using BSA and pig thyroglobulin (Tg) coupled to gold particles for electron microscopy, conjugated to fluorescein for conventional and confocal fluorescence microscopy, or radioiodinated for biochemical measurements. Incubations were carried out at 37 C. BSA and Tg coupled to gold particles were rapidly internalized from the culture medium and sequentially found in small vesicles and early endosomes and in late endosomes and lysosomes. Fluorescence microscope analyses revealed that the majority of cells forming reconstituted thyroid follicles are capable of internalizing BSA and Tg, but that Tg was more efficiently endocytosed than BSA. Using radioiodinated ligands, it was observed that the endocytosis of Tg was 10 times higher than that of BSA. The internalization of [125I]Tg was inhibited by increasing concentrations of unlabeled Tg. In contrast, endocytosis of 125I-labeled BSA was independent of the unlabeled BSA concentration. Experiments performed at 4 C indicated the presence of a basolateral membrane binding activity for [125I]Tg; the Tg concentration that reduced the binding of labeled Tg by 50% ranged from 4-6 microM. These data are evidence of a process of internalization of soluble molecules at the basolateral pole of thyrocytes, with BSA being internalized by fluid phase endocytosis and Tg by selective endocytosis. Our findings explain how serum albumin can enter thyroid follicles and disclose a new cellular handling and transport pathway of Tg. We propose that selective uptake of Tg operating on molecules secreted at the basolateral surface of thyrocytes could control the amount of Tg released in the circulation.

Asunto(s)

Endocitosis , Albúmina Sérica/fisiología , Tiroglobulina/fisiología , Glándula Tiroides/citología , Animales , Células Cultivadas , Técnicas Citológicas , Microscopía Electrónica , Porcinos , Glándula Tiroides/metabolismo

RESUMEN

The large family of steroid/thyroid hormone receptor (STR) genes has been extensively studied in vertebrates and insects but little information is available on it in more primitive organisms. All members possess a DNA binding domain of zinc fingers of the C2, C2 type. We have used the polymerase chain reaction with degenerate oligonucleotide primers covering this region to clone three distinct members of this family from the nematode Caenorhabditis elegans. All three belong to the retinoic acid receptor (RAR), thyroid hormone receptor subfamily of genes. The cDNA of one of these clones shows such a high homology to DHR3, an early ecdysone response gene found in Drosophila, and MHR3, identified in Manduca sexta, that we have termed it CHR3. Furthermore, the C-terminal portion of the deduced protein sequence shows a box containing eight identical amino acids among CHR3, DHR3, and MHR3 suggesting an identical specific ligand for these proteins. CNR8 shows homology to NAK1, and CNR14 has homology to both the RAR-gamma 1 gene and to another ecdysone response gene, E78A. Neither of the latter two cDNAs is a clear homologue of any known gene and each is distinctive. All of these genes are expressed varyingly in both larval and adult stages of nematode development as shown by Northern blot analyses. These data demonstrate that the STR family of genes is represented in a nematode whose ancestor appeared well before the branching that gave rise to the Arthropoda and Chordata.

Asunto(s)

Caenorhabditis elegans/genética , Familia de Multigenes , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Secuencia Conservada , Cisteína , Cartilla de ADN , Drosophila/genética , Biblioteca de Genes , Humanos , Manduca/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Esteroides/biosíntesis , Receptores de Hormona Tiroidea/biosíntesis , Homología de Secuencia de Aminoácido , Dedos de Zinc/genética

RESUMEN

Thyroglobulin (Tg) molecules stored in thyroid follicle lumens are heterogeneous in terms of iodine and hormone contents. It has been suggested that thyroid hormone is preferentially produced from the most highly iodinated Tg molecules and that thyrocytes are capable of selecting these molecules. The cellular localization as well as the molecular basis of such a selection process are not known. The present work was undertaken to determine whether there is selectivity at the step of endocytosis and, if not, to discover other possible mechanisms. Studies were conducted on reconstituted thyroid follicles (RTF) in culture. We compared the ability of thyrocytes to internalize Tg and an exogenous protein, BSA, which is neither iodinated nor glycosylated. To identify the protein, Tg and BSA were coupled to gold particles of different size and microinjected in a fixed ratio into the lumen of RTF. Neither of the two protein gold probes detected by transmission electron microscope bound at the cell surface, and both entered the cells at a similar rate and were concentrated in early endosomes. After 20 min, both Tg-G and BSA-G were segregated into distinct vacuolar structures. At 60 min, the intracellular content of BSA-G (mainly in prelysosomes and lysosomes) was 2- to 3-fold higher than that of Tg-G. At the same time, there was a marked reduction in the BSA-G/Tg-G ratio in the lumen. The differences between the Tg-G and BSA-G distribution patterns that were amplified in TSH-treated RTF are in keeping with a back-transfer of internalized Tg toward the lumen. The existence of a cell to lumen transport of previously endocytosed Tg was further documented using intralumenal [125I]Tg as a marker. RTF pulse labeled with tracer amounts of [125I]iodide were shortly incubated with TSH to induce [125I]Tg endocytosis, and the fate of internalized [125I] Tg was studied in a chase incubation period of up to 4 h. At 20 C, where the degradation of internalized Tg is blocked, we observed a time-dependent decrease in intracellular [125I]Tg and a corresponding increase in the lumenal [125I]Tg content. This cell to lumen [125I]Tg transfer was inhibited by primaquine. In conclusion, our data show that 1) the thyroid apical endocytic process does not exhibit selectivity for Tg; 2) the thyrocyte possesses a sorting machinery for endocytosed ligands; and 3) internalized Tg molecules can be recycled back to the follicular lumen.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Orgánulos/metabolismo , Tiroglobulina/farmacocinética , Glándula Tiroides/metabolismo , Animales , Oro , Membranas Intracelulares/metabolismo , Radioisótopos de Yodo , Microinyecciones , Microscopía Electrónica , Albúmina Sérica Bovina/farmacocinética , Glándula Tiroides/citología , Glándula Tiroides/ultraestructura , Distribución Tisular

RESUMEN

We have designed a new experimental system based on in vitro reconstituted thyroid follicles (RTF) to study the relative implication of macropinocytosis and micropinocytosis processes in the internalization of thyroglobulin (Tg). Thyrocytes cultured in the presence of TSH reorganize in histiotypic and functional follicles. Tg, which accumulates into the newly formed intrafollicular lumen (IL), was pulse labeled with [125I]iodide. Basal or TSH-activated Tg internalization, i.e. transfer from IL to cells, was assessed by measuring [125I]Tg in the cells and the IL; the IL fraction was collected after selective opening of lumina by a short treatment of RTF in a calcium-free medium. We used the ratio between cellular and IL labeled Tg contents as an endocytic index. TSH caused a very rapid increase in the cellular uptake of labeled Tg; the endocytic index increased by a factor of 4-8. The TSH effect was maximum after 15-20 min. TSH had no effect when the chase-incubation was performed at 4 C, but exhibited the same stimulatory action in terms of both time course and amplitude of action at 20 and 37 C. The macropinocytosis-related cellular structures, the pseudopods, were never observed in RTF maintained at 20 C; they were rare at 37 C and only found after 30 min of TSH treatment. At 20 as well as 37 C, the action of TSH on Tg endocytosis was concentration dependent in the range of 0.05-10 mU/ml. A fraction of Tg internalized by thyrocytes was found in coated vesicles. The labeled Tg content of purified coated vesicles varied with the temperature of the chase-incubation and was increased in TSH-treated RTF. Taken together, these data show that endocytosis of Tg by thyroid follicular cells in resting or moderately activated states does not proceed via the pseudopod formation-dependent mechanism, also termed macropinocytosis. Tg internalization would be related to what is referred as micropinocytosis and would involve a coated vesicle-dependent endocytic pathway.

Asunto(s)

Endocitosis/fisiología , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Animales , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Técnicas de Cultivo , Técnicas Histológicas , Métodos , Temperatura , Hormonas Tiroideas/metabolismo , Tirotropina/farmacología

RESUMEN

We have tried to characterize the intracellular compartments involved in the traffic of the thyroid prohormone thyroglobulin (Tg) from the site of storage, the follicular lumen, to the expected site(s) of proteolytic degradation, lysosomes. Electron microscope immunogold labeling with antibodies against Tg, cation-independent mannose-6-phosphate receptor (MPR), or arylsulfatase-A (ArS-A) was used to identify endocytic structures. The implication of these structures in the transport of Tg was analyzed by following the internalization and intracellular fate of Tg-colloidal gold complexes microinjected into the thyroid follicular lumen. Immunogold labeling was performed on ultrathin cryosections of intact pig tissue, in vitro reconstituted thyroid follicles (RTF), and isolated vesicles prepared by differential and isopycnic centrifugation. Microinjection experiments were carried out on RTF. Using double labeling for MPR and ArS-A, we characterized three types of structures: those slightly positive for MPR and ArS-A, those strongly positive for both markers, and those only positive for ArS-A. These compartments exhibited the properties of early endosomes (EE), late endosomes (LE), and lysosomes (L), respectively. Tg immunoreactivity was high in EE, low in LE, and undetectable in L. Similar morphological and immunochemical characteristics of EE, LE, and L were found in intact tissue, RTF, and isolated vesicles. Tg-gold complexes microinjected into the lumen of RTF were efficiently internalized within 5 min into structures with the appearance of EE. Sixty minutes after the injection, Tg-gold complexes were detected into LE and L. We present here the first direct experimental evidence for an involvement of endosomal compartments in the Tg internalization/degradation pathway. The data indicate that internalized Tg molecules are transported to EE and then transferred from EE to LE.

Asunto(s)

Orgánulos/metabolismo , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Animales , Oro , Técnicas Inmunológicas , Glándula Tiroides/citología , Factores de Tiempo , Distribución Tisular

RESUMEN

We have tried to identify iodinated thyroglobulin molecules in purified thyroid-coated vesicles to determined whether the internalization of the thyroid prohormone could proceed via a mechanism of receptor-mediated endocytosis. Coated vesicles isolated from pig thyroids by differential centrifugation and centrifugation on 2H2O-sucrose cushion were characterized by transmission electron microscopy and analyses of the polypeptide composition by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and Western blot using anti-clathrin heavy chain and anti-thyroglobulin antibodies. Clathrin and thyroglobulin (Tg) appeared as the two major components of the purified thyroid coated vesicles (TCV). Purified TCV fraction was homogeneous when analyzed by isopycnic centrifugation on 30% Percoll gradient. TCV had an apparent buoyant density of 1.035 g/ml. The presence of Tg molecules inside TCV was ascertained by (a) immunogold labeling on cryosections of TCV pellet and (b) identification by gel electrophoresis and radio-immunoassay of a definite fraction of Tg (3-5% of total protein) in TCV treated by Triton X-100. The detergent-treated TCV also contained protein-bound iodine: 0.5-0.7 micrograms of iodine/mg protein. Pulse-chase experiments on in vitro reconstituted thyroid follicles have been used to further document the presence of iodinated Tg molecules in coated vesicles. TCV were isolated from reconstituted thyroid follicles previously labeled with [125I]iodide to radioiodinate Tg of the follicular lumen (the pre-endocytotic compartment) and incubated with or without thyrotropin or dibutyryl cyclic AMP to activate intraluminal 125I-Tg endocytosis. Autoradiographic analyses revealed the presence of 125I-Tg in purified TCV and Triton X-100-treated TCV. 125I-Tg present in TCV represented 1-2% of the total intracellular protein-bound radioactivity. Thyrotropin and dibutyryl cyclic AMP increased 2-3-fold the 125I-Tg content of TCV. Our results clearly show that iodinated Tg, the molecular form of the thyroid prohormone known to be internalized, is present into TCV. The data suggest that coated vesicles are involved in the uptake and transport of Tg from the follicular lumen to the lysosomal compartment and therefore, that the internalization of Tg could proceed, at least for a part, via a mechanism of receptor-mediated endocytosis.

Asunto(s)

Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis , Precursores de Proteínas/metabolismo , Receptores de Superficie Celular , Receptores de Hormona Tiroidea/metabolismo , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Animales , Bucladesina/farmacología , Fraccionamiento Celular , Células Cultivadas , Centrifugación por Gradiente de Densidad , Invaginaciones Cubiertas de la Membrana Celular/efectos de los fármacos , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Microscopía Electrónica , Microscopía Inmunoelectrónica , Povidona , Dióxido de Silicio , Porcinos , Tiroglobulina/aislamiento & purificación , Glándula Tiroides/ultraestructura , Tirotropina/farmacología , Ultracentrifugación

RESUMEN

Thyroid gland from normal and hormonally influenced rats (by application of exogenous Triiodothyronine and Thyroid stimulating Hormone), were investigated by means of classical electron microscopy. Dense granules, micropinocytotic and macropinocytotic structures were observed similarly as was already published. Triiodothyronine and Thyroglobulin were localized by affinity cytochemistry on ultrathin cryosections from normal rat thyroid gland. These method showed, that particular endocytotic and exocytotic structures can't be distinguish only by simple morphology and that functional status of these structures can be evaluated only by detection of contained molecules. Triioodthyronine was observed by affinity cytochemistry in multivesicular bodies and in lateral intercellular spaces. That findings indicate, that Triiodothyronine is internalized (transcytosed) by Receptor-mediated endocytosis. Affinity cytochemistry is useful for study of triiodothyronine and thyroglobulin movement in the thyroid gland.

Asunto(s)

Glándula Tiroides/ultraestructura , Animales , Ratas , Ratas Endogámicas Lew , Pruebas de Función de la Tiroides , Glándula Tiroides/metabolismo