Orlie Levy

The mammary gland iodide transporter is expressed during lactation and in breast cancer

The sodium/iodide symporter mediates active iodide transport in both healthy and cancerous thyroid tissue. By exploiting this activity, radioiodide has been used for decades with considerable success in the detection and treatment of thyroid cancer. Here we show that a specialized form of the sodium/iodide symporter in the mammary gland mediates active iodide transport in healthy lactating (but not in nonlactating) mammary gland and in mammary tumors. In addition to characterizing the hormonal regulation of the mammary gland sodium/iodide symporter, we demonstrate by scintigraphy that mammary adenocarcinomas in transgenic mice bearing Ras or Neu oncogenes actively accumulate iodide by this symporter in vivo. Moreover, more than 80% of the human breast cancer samples we analyzed by immunohistochemistry expressed the symporter, compared with none of the normal (nonlactating) samples from reductive mammoplasties. These results indicate that the mammary gland sodium/iodide symporter may be an essential breast cancer marker and that radioiodide should be studied as a possible option in the diagnosis and treatment of breast cancer.

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.

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.

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.

The Na+/I− Symporter (NIS): Recent Advances

The Na+/I− symporter (NIS) catalyzes the accumulation of iodide into thyroid cells, an essential step in the biosynthesis of thyroid hormones. As a result of the isolation of the rat NIS cDNA, steadfast advances in the study of NIS at the molecular level have resulted in the following accomplishments: generation of high-affinity anti-NIS antibodies, elucidation of NIS stoichiometry and specificity by electrophysiological analysis, biochemical and immunological experimental testing of the proposed NIS secondary structure model, monitoring the regulation of NIS protein expression by thyroid stimulating hormone and iodide, characterization of the rat NIS gene promoter, isolation of the cDNA clone encoding human NIS and subsequent determination of human NIS genomic organization, description of NIS mutations in patients with congenital lack of iodide transport, and the molecular identification of NIS in extrathyroidal tissues.

Structure and function of the thyroid iodide transporter and its implications for thyroid disease

The thyroid iodide (I−) transporter, a Na+/I− symporter (NIS), is an intrinsic 618-amino-acid membrane glycoprotein that catalyzes the accumulation of I− into thyrocytes, an essential step in the biosynthesis of thyroid hormones. Remarkable progress has recently been achieved in the study of NIS at the molecular level as a result of the isolation of the cDNA encoding rat NIS. Among the main accomplishments are the isolation of the cDNA clone encoding human NIS, electrophysiological analysis and elucidation of the mechanism, stoichiometry, and specificity of rat NIS, generation of anti-rat NIS antibodies for use in NIS characterization, proposal and experimental testing of NIS secondary structure models, analysis of the regulation of NIS expression by thyroid-stimulating hormone and I−, characterization of the regulation of the rat NIS gene, elucidation of the genomic organization of human NIS, identification of an NIS mutation in a case of congenital lack of I“ transport, and detection of anti-NIS autoantibodies in thyroid autoimmune disease.

Chapter 15 The mediator of thyroidal iodide accumulation: The sodium/iodide symporter

Publisher Summary The Na+/I- symporter (NIS) is a key plasma membrane protein that catalyzes the active accumulation of iodide (I-) in the thyroid gland, that is, the first and critical rate-limiting step in the biosynthesis of the thyroid hormones. NIS is located in the basolateral membrane of the hormone-producing thyroid follicular cells or thyrocytes. NIS is one of at least four major proteins that appear to be distinctively thyroidal, the other three being Tg, TPO and the TSH receptor. Each of these four proteins mediates a crucial step in thyroid hormogenesis. Each of them also plays an important role in thyroid function. They are also considered thyroid-specific markers. This chapter presents the rat NIS cDNA clone and discusses a secondary structure model for the NIS molecule. The product of the NIS cDNA clone is sufficient to elicit Na+/I- symport activity in both oocytes and mammalian cells, suggesting that NIS probably functions as a single subunit or as an oligomer of identical subunits. The role of NIS as the mediator of I- accumulation is consistent with its placing alongside other Na+-dependent anion transporters based on clustering relationships between its deduced amino acid sequence and the deduced sequences of other transporters. A comparison of the predicted amino acid sequence of NIS with those of other cloned Na+-dependent cotransporters revealed the highest degree of homology.