Nancy Carrasco

Thyroid Na+/I− Symporter MECHANISM, STOICHIOMETRY, AND SPECIFICITY

The rat thyroid Na+/I− symporter (NIS) was expressed inXenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ ≫ Li+ ≫ H+). The apparent affinity constants for Na+ and I− were 28 ± 3 mm and 33 ± 9 μm, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I−, ClO3 −, SCN−, SeCN−, NO3 −, Br−, BF4 −, IO4 −, BrO3 −, but perchlorate (ClO4 −) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (∼35% of maximum substrate-induced current) with an apparent Na+ affinity of 74 ± 14 mm and a Hill coefficient (n) of 1. In response to step voltage changes, NIS exhibited current transients that relaxed with a time constant of 8–14 ms. Presteady-state charge movements (integral of the current transients) versus voltage relations obey a Boltzmann relation. The voltage for half-maximal charge translocation (V 0.5) was −15 ± 3 mV, and the apparent valence of the movable charge was 1. Total charge was insensitive to [Na+] o , but V 0.5 shifted to more negative potentials as [Na+] o was reduced. NIS charge movements are attributed to the conformational changes of the empty transporter within the membrane electric field. The turnover rate of NIS was ≥22 s−1 in the Na+ uniport mode and ≥36 s−1 in the Na+/I− cotransport mode. Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a ∼2.5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mechanism in which the binding of Na+ to NIS occurs first.

Iodide transport in the thyroid gland

III. Nature of the I transport process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 A. I accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 B. I efflux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

The Paired-Domain Transcription Factor Pax8 Binds to the Upstream Enhancer of the Rat Sodium/Iodide Symporter Gene and Participates in Both Thyroid-Specific and Cyclic-AMP-Dependent Transcription

ABSTRACT The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides −2264 and −2495 in the 5′-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site.

N-linked Glycosylation of the Thyroid Na+/I− Symporter (NIS) IMPLICATIONS FOR ITS SECONDARY STRUCTURE MODEL

The Na+/I−symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active accumulation of I− into thyroid cells, was identified and characterized at the molecular level in our laboratory (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458–460). Because mature NIS is highly glycosylated, it migrates in SDS-polyacrylamide gel electrophoresis as a broad polypeptide of higher molecular mass (∼90–110 kDa) than nonglycosylated NIS (∼50 kDa). Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putativeN-linked glycosylation consensus sequences of NIS with glutamine and assessed the effects of the mutations on function and stability of NIS in COS cells. All mutants were active and displayed 50–90% of wild-type NIS activity, including the completely nonglycosylated triple mutant. This demonstrates that to a considerable extent, function and stability of NIS are preserved in the partial or even total absence of N-linked glycosylation. We also found that Asn225 is glycosylated, thus proving that the hydrophilic loop that contains this amino acid residue faces the extracellular milieu rather than the cytosol as previously suggested. We demonstrated that the NH2 terminus faces extracellularly as well. A new secondary structure model consistent with these findings is proposed.

Advances in Na+/I− symporter (NIS) research in the thyroid and beyond

The Na(+)/I(-) symporter (NIS) is a plasma membrane glycoprotein that mediates active iodide uptake in the thyroid-the essential first step in thyroid hormone biosynthesis-and in other tissues, such as salivary and lactating mammary glands. Thyroidal radioiodide uptake has been used for over 60 years in the diagnosis and effective treatment of thyroid cancer and other diseases. However, the NIS cDNA was only isolated in 1996 by expression cloning in Xenopus laevis oocytes, marking the beginning of the molecular characterization of NIS and the study of its regulation, both in the thyroid and other tissues. One of the most exciting current areas of NIS research-radioiodide treatment of extrathyroidal cancers-was launched by the discovery of functional expression of endogenous NIS in breast cancer and by the ectopic transfer of the NIS gene into otherwise non NIS-expressing cancers. This review summarizes the main findings in NIS research, emphasizing the most recent developments.

Moderate doses of iodide in vivo inhibit cell proliferation and the expression of thyroperoxidase and Na+/I− symporter mRNAs in dog thyroid

The function and the growth of adult thyroid gland is controlled by the opposite actions of thyrotropin (TSH) and iodide, the main substrate of the gland. Iodide deprivation leads to stimulation of the thyroid, improving the efficiency of iodide transport for hormone biosynthesis. We have investigated cell proliferation and thyroid specific gene expression 24 and 48 h after administering KI to dogs previously treated with goitrogens and perchlorate. In the hypothyroid dogs T3 and T4 serum levels decreased from 53 +/- 4 to < 30 ng/dl and from 1.6 +/- 0.6 to < 1 microg/dl respectively; TSH concentration increased from 0.16 +/- 0.02 to 2.7 +/- 0.4 ng/ml. After a 24 h moderate KI treatment (300 microg KI/dog of +/- 10 kg) serum T3 concentrations rose higher than the initial normal values, while T4 concentrations increased to reach values equivalent to the normal level. The high TSH concentration did not change significantly. The hyperplasia of the chronically stimulated thyroid resulting from goitrogens/NaClO4 treatment was not modified by this short term treatment with KI. In contrast, KI decreased the weight of the total gland and the level of cell proliferation, as determined by the fraction of cells incorporating BrdU. The effect of acute administration of KI on the expression of four major thyroid genes, the TSH receptor (TSHr), thyroglobulin (Tg), thyroperoxidase (TPO), and Na+/I- symporter (NIS) was analyzed by Northern blot. Tg, TPO and NIS mRNA expressions were up-regulated by chronic stimulation. The expression of the mRNAs of TSHr and Tg did not significantly differ between hyperstimulated and KI-treated dogs while TPO and NIS mRNA expression decreased after a 48 h KI treatment. TPO and NIS are therefore the only of these four genes whose expression is acutely modulated by iodide in vivo. Under TSH stimulation low doses of iodide resulted in: (1) decreased cell proliferation, (2) reestablished synthesis and secretion of thyroid hormones, (3) diminished TPO and NIS mRNA expression. Notably low doses of iodide under the same conditions had no effect on Tg and TSHr mRNA expression.

Identification of a structural requirement for thyroid Na+/I− symporter (NIS) function from analysis of a mutation that causes human congenital hypothyroidism

Patients with congenital lack of I− transport do not accumulate I− in their thyroids, often resulting in severe hypothyroidism. A single amino acid substitution in the thyroid Na+/I− symporter (NIS), proline replacing threonine at position 354 (T354P), was recently identified as the cause of this condition in two independent patients [1, 2]. Here we report that the lack of I− transport activity in T354P NIS generated by site‐directed mutagenesis, is not due to a structural change induced by proline, but rather to the absence of a hydroxyl group at the β‐carbon of the amino acid residue at position 354. Hence, this hydroxyl group is essential for NIS function.

Journey of the iodide transporter NIS: from its molecular identification to its clinical role in cancer

The Na+/I- symporter (NIS) is an intrinsic plasma membrane protein that mediates the active transport of I- in the thyroid, lactating mammary gland, stomach and salivary glands. The presence of NIS in the thyroid is exploited in diagnostic scintigraphic imaging and radioiodide therapy in thyroid cancer. The continued rapid progress in NIS research (aimed at the elucidation of the Na+-dependent I- transport mechanism, the analysis of NIS structure-function relations and the study of the tissue-specific regulation of NIS at all levels), holds potentially far-reaching medical applications beyond thyroid disease, in breast cancer and malignancies in other tissues.

Molecular Study of the Sodium–Iodide Symporter (NIS): A New Field in Thyroidology

The active transport of iodide into the thyroid is mediated by the Na(+)-I- symporter (NIS), an intrinsic membrane protein. NIS plays key roles in thyroid pathophysiology as the route by which I- reaches the gland for thyroid hormone biosynthesis, and as a means for diagnostic scintigraphic imaging and for radioiodide therapy in thyroid cancer. The molecular characterization of NIS started with the isolation in 1996 of a cDNA encoding rat NIS, and has subsequently led to a virtually new field in thyroidology. The research reviewed in this article clearly has far-reaching implications in the areas of structure/function of transport proteins, thyroid pathophysiology, hormone action mechanisms, cell differentiation and cancer.

Expression of the Na+/l-symporter (NIS) is markedly decreased or absent in gastric cancer and intestinal metaplastic mucosa of Barrett esophagus

BackgroundThe sodium/iodide symporter (NIS) is a plasma membrane glycoprotein that mediates iodide (I-) transport in the thyroid, lactating breast, salivary glands, and stomach. Whereas NIS expression and regulation have been extensively investigated in healthy and neoplastic thyroid and breast tissues, little is known about NIS expression and function along the healthy and diseased gastrointestinal tract.MethodsThus, we investigated NIS expression by immunohistochemical analysis in 155 gastrointestinal tissue samples and by immunoblot analysis in 17 gastric tumors from 83 patients.ResultsRegarding the healthy Gl tract, we observed NIS expression exclusively in the basolateral region of the gastric mucin-producing epithelial cells. In gastritis, positive NIS staining was observed in these cells both in the presence and absence of Helicobacter pylori. Significantly, NIS expression was absent in gastric cancer, independently of its histological type. Only focal faint NIS expression was detected in the direct vicinity of gastric tumors, i.e., in the histologically intact mucosa, the expression becoming gradually stronger and linear farther away from the tumor. Barrett mucosa with junctional and fundic-type columnar metaplasia displayed positive NIS staining, whereas Barrett mucosa with intestinal metaplasia was negative. NIS staining was also absent in intestinalized gastric polyps.ConclusionThat NIS expression is markedly decreased or absent in case of intestinalization or malignant transformation of the gastric mucosa suggests that NIS may prove to be a significant tumor marker in the diagnosis and prognosis of gastric malignancies and also precancerous lesions such as Barrett mucosa, thus extending the medical significance of NIS beyond thyroid disease.