RESUMO
Expression of sodium/iodide symporter (NIS) by thyroid epithelial cells is primarily regulated by TSH, which acts at the level of NIS gene transcription. Knowledge of the mechanisms governing NIS expression mainly comes from studies of rat thyroid-derived cell lines forming cell monolayers. In this study we investigated the impact of the three-dimensional organization of thyroid cells into follicles on the regulation of NIS expression. We used porcine thyrocytes in primary culture that, depending on cell density and the moment TSH is added, either predominantly form a cell monolayer (CM) or reconstitute thyroid follicles (RTF). NIS expression analyzed at transcript and protein levels was remarkably high in RTF compared with CM. Cells forming RTF were NIS positive, whereas in CM, NIS was only detected in the limited number of cells forming follicle-like structures. When thyrocytes were cultured at increasing cell density to obtain a gradual shift from CM to RTF, the progressive increase in the proportion of cells enrolled in RTF was accompanied by a parallel increase in NIS expression. Other TSH-regulated genes, thyroperoxidase, Na(+),K(+)-adenosine triphosphatase alpha-subunit, and thyroglobulin, were expressed at similar levels whatever the organization of thyrocytes in culture. The transcription factor, Pax-8, was equally expressed in NIS-negative CM and NIS-positive RTF. We show that TSH highly activates NIS expression only when thyrocytes have undergone histiotypic morphogenesis. This finding suggests that TSH activation of NIS gene transcription might involve, in addition to Pax-8, a regulatory factor(s) whose synthesis and/or activity are triggered by cell-cell interaction(s) occurring in the course of folliculogenesis.
Assuntos
Regulação da Expressão Gênica , Simportadores/genética , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Animais , Células Cultivadas , Suínos , Simportadores/análise , Tireotropina/farmacologiaRESUMO
The sodium/iodide symporter (NIS) is a membrane protein mediating the active transport of iodide into the thyroid gland. NIS, expressed by human, rat, and mouse thyrocytes, is encoded by a single transcript. We identified NIS mRNA species of 3.5 and 3 kb in porcine thyrocytes. Because porcine thyrocytes in primary culture is a widely used experimental system for thyroid iodide metabolism, we further examined the origin and the function of the porcine NIS (pNIS) transcripts. We generated a porcine thyroid cDNA library from which four different clones, pNIS-D, F, J, and Delta J were isolated. pNIS-D encodes a protein of 643 amino acids highly homologous to the human, rat, and mouse NIS. pNIS-F and J differ from each other and from pNIS-D in their C-terminal part. pNIS-Delta J lacks a six-amino-acid segment within the putative transmembrane domain 10. Transiently expressed in Cos-7 cells, the four pNIS-cDNAs led to the synthesis of proteins targeted at the plasma membrane and conferred perchlorate-sensitive iodide uptake activities to Cos-7 cells, except pNIS-Delta J, which was devoid of activity. PNIS-D probably derives from the 3.5-kb transcript and pNIS-F, J, and Delta J from the 3-kb transcript. The relative abundance of pNIS-D, F, and J transcripts in porcine thyrocytes was about 60%, 35%, and 5%, respectively; the Delta J transcript was not present in detectable amount. By comparing porcine NIS genomic and cDNA sequences, splice donor and acceptor sites accounting for the generation of pNIS-F, J, and Delta J transcripts were identified. None of the combinations of alternative splice sites found in the pig was present in the human, rat or mouse NIS gene. Our data show that porcine NIS gene, contrary to the NIS gene from other species, gives rise to splice variants leading to three active and one inactive NIS proteins.
Assuntos
Processamento Alternativo , Expressão Gênica , Suínos/genética , Simportadores/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células COS , Células Cultivadas , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/química , Técnica Indireta de Fluorescência para Anticorpo , Biblioteca Gênica , Humanos , Iodetos/metabolismo , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Alinhamento de Sequência , Especificidade da Espécie , Simportadores/química , Glândula Tireoide/química , TransfecçãoRESUMO
The gene mutated in Pendred syndrome (PDS), the PDS gene, is expressed in the inner ear, kidney, and thyroid. It encodes a membrane protein named pendrin that is endowed with the function of anion transporter or exchanger. It has been postulated that in the thyroid pendrin could participate in the transport of iodide from the cell to the lumen of follicles. We generated antipeptide antibodies directed against the C- terminal sequence of human pendrin 1) to characterize the protein expressed in the human thyroid, and 2) to analyze its expression level in relation to the functional activity of thyroid tissue. In denaturing conditions, a single molecular species of 110-115 kDa was identified in human thyroid membrane fractions. After treatment of thyroid membranes with N-glycosidase F, pendrin had an apparent molecular mass of 85 kDa. Analyzed by ultracentrifugation on sucrose gradient in nondenaturing conditions, pendrin sedimented as a main 120- to 140-kDa component. Pendrin was assayed by semiquantitative Western blot in thyroid membrane fractions from 25 hyper- or hypofunctioning tumors and paired normal tissue samples. Pendrin was increased 2-fold in toxic adenomas, was not significantly altered in follicular adenoma, and was decreased, on the average, by 35% in papillary carcinomas compared with levels in paired normal tissue. The variations in the pendrin tissue content and PDS transcript levels, assayed by RT-PCR on duplicate samples of the same tumors, were similar. In conclusion, we show that pendrin expressed by the human thyroid gland is a mainly monomeric glycoprotein and that the level of expression of pendrin, although somewhat related, only moderately varied with the functional status of the thyroid tissue.
Assuntos
Carcinoma Papilar/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adulto , Carcinoma Papilar/fisiopatologia , Feminino , Bócio/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Valores de Referência , Transportadores de Sulfato , Simportadores/genética , Síndrome , Glândula Tireoide/citologia , Neoplasias da Glândula Tireoide/fisiopatologiaRESUMO
The uptake of iodide by epithelial thyroid cells requires the expression of a specific transporter, the Na(+)/I(-) symporter, NIS. Benign and malignant thyroid tumors of epithelial origin show a decrease up to a loss of iodide uptake activity. Previous studies of the human NIS (hNIS) gene expression in these tumors, based on the amplification of transcripts and/or immunohistochemical detection of the protein, have yielded divergent data; hNIS expression was found either increased or decreased. To get a new and integrated view of the alterations of hNIS expression in hypofunctioning thyroid tumors, we performed investigations of hNIS transcript and hNIS protein levels on the same tumors and paired normal tissue samples. HNIS, identified as a 75- to 80-kd species, was present in all normal tissue samples from euthyroid patients, but was undetectable, even at high membrane protein input, in all benign and malignant hypofunctioning thyroid tumors. By contrast, approximately 50% of tumors contained hNIS transcripts. This dissociation between transcript and protein levels was not found for the transcript and protein encoded by the PDS gene assayed in the same tumors. The hNIS transcript-positive tumors contained small amounts of low-molecular mass hNIS-immunoreactive species identified as nonglycosylated hNIS. Tumors containing the nonmature form of hNIS exhibited a predominant intracellular immunolabeling. In conclusion, our data show that benign and malignant hypofunctioning thyroid tumors either no longer express hNIS protein or express only a very low amount of nonglycosylated hNIS and indicate that the impairment of hNIS gene expression might result from alterations at both transcriptional and posttranscriptional levels.