The Sodium/Iodide Symporter(NIS): Characterization Regulation and Medical Significance

http://www.100md.com 《内分泌进展》2003年第1期

     Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461orrespondence and requests for reprints to: Nancy;, http://www.100md.com

    Correspondence: Address all c;, http://www.100md.com

    Abstract;, http://www.100md.com

    The Na⁺/I^- symporter (NIS) is an integral plasma membrane glycoprotein that mediates active I^- transport into the thyroid follicular cells, the first step in thyroid hormone biosynthesis. NIS-mediated thyroidal I^- transport from the bloodstream to the colloid is a vectorial process made possible by the selective targeting of NIS to the basolateral membrane. NIS also mediates active I^- transport in other tissues, including salivary glands, gastric mucosa, and lactating mammary gland, in which it translocates I^- into the milk for thyroid hormone biosynthesis by the nursing newborn. NIS provides the basis for the effective diagnostic and therapeutic management of thyroid cancer and its metastases with radioiodide. NIS research has proceeded at an astounding pace after the 1996 isolation of the rat NIS cDNA, comprising the elucidation of NIS secondary structure and topology, biogenesis and posttranslational modifications, transcriptional and posttranscriptional regulation, electrophysiological analysis, isolation of the human NIS cDNA, and determination of the human NIS genomic organization. Clinically related topics include the analysis of congenital I^- transport defect-causing NIS mutations and the role of NIS in thyroid cancer. NIS has been transduced into various kinds of cancer cells to render them susceptible to destruction with radioiodide. Most dramatically, the discovery of endogenous NIS expression in more than 80% of human breast cancer samples has raised the possibility that radioiodide may be a valuable novel tool in breast cancer diagnosis and treatment.

    I. Introduction and Background9m21+v2, 百拇医药

    II. Molecular Characterization of NIS9m21+v2, 百拇医药

    A. Summary of the molecular characteristics of NIS9m21+v2, 百拇医药

    B. NIS protein family9m21+v2, 百拇医药

    C. The road to NIS characterization9m21+v2, 百拇医药

    III. Transcriptional Regulation of NIS9m21+v2, 百拇医药

    IV. Regulation of NIS Expression and Function9m21+v2, 百拇医药

    A. TSH9m21+v2, 百拇医药

    B. Posttranscriptional regulation of NIS9m21+v2, 百拇医药

    C. Regulation of NIS activity by I^-9m21+v2, 百拇医药

    D. Effect of cytokines on NIS9m21+v2, 百拇医药

    E. Tg9m21+v2, 百拇医药

    F. Estradiol9m21+v2, 百拇医药

    V. Signal Transduction9m21+v2, 百拇医药

    VI. Extrathyroidal NIS Expression9m21+v2, 百拇医药

    A. Mammary gland NIS (mg-NIS)9m21+v2, 百拇医药

    B. NIS in the gastrointestinal tract

    C. Placental NIS#[&-h2], 百拇医药

    D. Kidney NIS#[&-h2], 百拇医药

    VII. Congenital ITD due to NIS Mutations#[&-h2], 百拇医药

    VIII. NIS in Autoimmune Thyroid Disease (AITD)#[&-h2], 百拇医药

    IX. NIS and Cancer#[&-h2], 百拇医药

    A. Thyroid cancer#[&-h2], 百拇医药

    B. Breast cancer#[&-h2], 百拇医药

    X. NIS in Gene Transfer#[&-h2], 百拇医药

    XI. Concluding Remarks#[&-h2], 百拇医药

    I. Introduction and Background#[&-h2], 百拇医药

    THE Na⁺/I^- SYMPORTER (NIS) is an integral plasma membrane glycoprotein most commonly studied and discussed in connection with the thyroid gland, in which NIS mediates the active transport of I^- into the thyroid follicular cells as the crucial first step for thyroid hormone biosynthesis. The thyroid hormones T₃ and T₄ are the only iodine-contining hormones in vertebrates. Because I^- is an essential constituent of T₃ and T₄, both thyroid function and its systemic ramifications depend on an adequate supply of I^- to the gland (1). This supply, in turn, depends on sufficient dietary intake of I^- and proper NIS function. NIS also mediates active I^- transport in other tissues, including salivary glands, gastric mucosa, and lactating mammary gland. Whereas the functional significance of NIS in the gastric mucosa and salivary glands is unknown, in the lactating mammary gland NIS mediates the translocation of I^- into the milk, making this anion available for the nursing newborn to biosynthesize his/her own thyroid hormones (1).

    The ability of the thyroid to accumulate I^- via NIS has long provided the basis for diagnostic scintigraphic imaging of the thyroid with radioiodide and has served as an effective means for therapeutic doses of radioiodide to target and destroy hyperfunctioning thyroid tissue, such as in Graves’ disease and I^--transporting thyroid cancer and its metastases (2). Therefore, the study of NIS is of great relevance to thyroid pathophysiology. Nevertheless, no molecular information on NIS was available until 1996, when our group (3), by expression cloning in Xenopus laevis oocytes, isolated a cDNA encoding rat NIS (rNIS). This development, a major breakthrough in the study of I^- transport processes and thyroid physiology, marked the beginning of the molecular characterization of NIS.@2, 百拇医药

    NIS research has since proceeded at an astounding pace with a wide variety of approaches and techniques, leading to numerous reports (see entire reference section) and reviews (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) in just the last few years. NIS secondary structure and topology have been experimentally tested; the biogenesis and posttranslational modifications of NIS have been examined; a thorough electrophysiological analysis of NIS has been conducted; the cDNA encoding human NIS (hNIS) has been isolated; the genomic organization of hNIS has been elucidated; the regulation of NIS by TSH, I^-, and other modulators has been analyzed; the regulation of NIS transcription has been studied; spontaneous NIS mutations have been identified as causes of congenital I^- transport defect (ITD) that results in hypothyroidism, and the molecular characterization of the mutant NIS proteins has yielded relevant structure/function information; the roles of NIS in thyroid cancer and autoimmune thyroid disease have been examined, and the expression and regulation of NIS in extrathyroidal tissues have been investigated. Interestingly, NIS has been found to be differently regulated and subjected to distinct posttranslational modifications in each tissue in which it is expressed. This disproves the previously held view of NIS as a thyroid-specific protein, such as thyroglobulin (Tg) and thyroid peroxidase (TPO), presumably not expressed in any other tissue.

    A significant recent finding on NIS is our report (14) demonstrating that more than 80% of the human breast cancer samples studied expressed NIS, whereas none of the normal samples did. These observations suggest that NIS expression in mammary adenocarcinomas and/or their metastases may be a valuable diagnostic and/or prognostic marker in breast cancer and raise the possibility that radioiodide may prove to be a valuable agent in the diagnosis and treatment of breast cancer. Radioiodide therapy has been used for more than 60 yr in thyroid cancer, particularly to destroy micrometastases after thyroidectomy (2, 15). This therapy is specifically targeted, inexpensive, readily and widely available, and causes only a few mild and infrequent side effects. Therefore, if radioiodide proves effective in breast cancer and/or its metastases, it would represent a highly significant advance in the management of the most lethal malignancy in women. Additional indications of the potential value of NIS and radioiodide in cancer are highly promising efforts to use gene therapy techniques to transduce and express NIS in cancer cells from a variety of tissues to render them susceptible to destruction with radioiodide.

    The ability of thyroid follicular cells to concentrate I^- was first reported as early as 1896 (16). The thyroid gland was found to concentrate I^- by a factor of 20–40 with respect to the plasma under physiological conditions. Hence, the existence of a thyroid I^- transporter was inferred, and some of its properties, along with the thyroid hormone biosynthetic pathway, were elucidated over the years (see Ref. 1 , and Refs. 17, 18, 19 for reviews). Briefly (Fig. 1), NIS-mediated I^- accumulation in the thyroid is an active transport process that occurs at the basolateral plasma membrane of the thyroid follicular cells against the I^- electrochemical gradient, stimulated by TSH and inhibitable by the well-known classic competitive inhibitors thiocyanate (SCN^-) and perchlorate (ClO₄^-). I^- is then translocated from the cytoplasm across the apical plasma membrane toward the colloid in a process called I^- efflux, which has been proposed to be mediated by pendrin (a Cl^-/I^- transporter; Ref. 20), and recently, by the apical I^- transporter (AIT; Ref. 20A ). In a complex reaction at the cell-colloid interface, called organification of I^- and catalyzed by TPO, I^- is oxidized and incorporated into some tyrosyl residues within the Tg molecule, leading to the subsequent coupling of iodotyrosine residues (Fig. 1). The term organification refers to the incorporation of I^- into organic molecules, as opposed to nonincorporated, inorganic, or free I^-. The I^- organification reaction can be pharmacologically blocked by 6-n-propyl-2-thiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI). Iodinated Tg is stored extracellularly in the colloid. In response to demand for thyroid hormones, phagolysosomal hydrolysis of endocytosed iodinated Tg ensues. T₃ and T₄ are secreted into the bloodstream, and nonsecreted iodotyrosines are metabolized to tyrosine and I^-, a reaction catalyzed by the microsomal enzyme iodotyrosine dehalogenase. This process facilitates reutilization of the remaining I^-. All of these steps, like NIS-mediated I^- uptake, are stimulated by TSH. In contrast, I^- accumulation in extrathyroidal tissues is not regulated by TSH (1).

    fig.ommittedig, 百拇医药

    Figure 1. Schematic representation of the biosynthetic pathway of thyroid hormones T₃ and T₄ in the thyroid follicular cell. Thyroid follicles are comprised of a layer of epithelial cells surrounding the colloid. The basolateral surface of the cell is shown on the left side of the figure and the apical surface on the right. Circle, Active accumulation of I^-, mediated by the NIS; triangle, Na⁺/K⁺ ATPase; square, TSH receptor; diamond, adenylate cyclase; ellipse, G protein; cylinder, I^- efflux toward the colloid; TPO, TPO-catalyzed organification of I^-; arrows pointing from the apical to the basolateral side indicate endocytosis of iodinated Tg, followed by phagolysosomal hydrolysis of endocytosed iodinated Tg and secretion of both thyroid hormones. AIT, Apical I^- transporter.ig, 百拇医药

    The significance of thyroid NIS becomes more apparent when one considers that I^- is scarce in the environment. Endemic goiter and cretinism caused primarily by insufficient dietary supply of I^- remain a major health problem in many parts of the world, affecting millions of people (21, 22). I^- deficiency still often leads to various degrees of impaired brain development (Table 1). These public health problems could conceivably be solved relatively easily by ensuring that all table salt consumed in the affected areas is iodized, as has been done in many countries. However, the sociopolitical realities of the affected regions have prevented measures such as this one from being implemented, at great human cost. Still, this situation dramatizes the health value of I^- as a nutrient and the consequences to society of its environmental scarcity. The present article provides an overview of the most recent developments in NIS research, both thyroidal and extrathyroidal.

    fig.ommitted4$.?chk, http://www.100md.com

    Table 1. Epidemiological impact of iodine deficiency disorders (IDD)4$.?chk, http://www.100md.com

    II. Molecular Characterization of NIS4$.?chk, http://www.100md.com

    A. Summary of the molecular characteristics of NIS4$.?chk, http://www.100md.com

    Membrane proteins in general and membrane transport proteins in particular have not been amenable to high-resolution structure determinations by traditional means because of their hydrophobicity and, in many instances, because of their metastable nature. Structures of membrane transporters at atomic resolution have only been obtained in a handful of instances (some examples are found in Refs. 23, 24, 25, 26, 27). In the absence of nucleotide and protein sequence information, the detailed molecular characterization of NIS started in 1996 when our group (3) isolated the cDNA encoding rNIS by expression cloning in X. laevis oocytes, using cDNA libraries derived from FRTL-5 cells (a highly functional rat thyroid-derived cell line). Another major development in the molecular characterization of NIS was the generation, also in our laboratory (28), of a high affinity (K_d ~ 1 nM) site-directed polyclonal anti-NIS antibody (Ab) against the last 16 amino acid residues of the COOH terminus of the protein (see Section II.C.1). Subsequently and independently, another site-directed Ab against the same COOH terminus segment of NIS was generated by Paire et al. (29). On the basis of the cloned cDNA, we determined that rNIS was a protein of 618 amino acids (with a relative molecular mass of 65,196). The hydropathic profile and initial secondary structure predictions of the protein suggested an intrinsic membrane protein with 12 putative transmembrane segments (3, 4). We initially placed the NH₂ terminus on the cytoplasmic side, given the absence of a signal sequence. The COOH terminus, which was also predicted to be on the cytoplasmic side, was found to contain a large hydrophilic region of approximately 70 amino acids, within which several potential phosphorylation consensus sequences of the molecule were located. This 12-transmembrane-segment model has since been experimentally tested by a variety of techniques and revised according to the results.

    Our current secondary structure model proposes 13 transmembrane segments with the NH₂ terminus facing extracellularly and the COOH terminus facing intracellularly (Fig. 2). Immunofluorescence experiments in our laboratory (30, 31) have confirmed these predicted orientations for both termini, as explained below. Three potential Asn-glycosylation sites were identified in the deduced amino acid sequence at positions 225, 485, and 497 (see Section II.C.1). The predicted length of the 13 transmembrane segments ranges from 20–28 amino acid residues, except for transmembrane segment V, which contains 18 residues. Only three charged residues are predicted to lie within transmembrane segments, namely Asp 16 in transmembrane segment I, Glu 79 in transmembrane segment II, and Arg 208 in transmembrane segment VI. Of a total of eight Trp residues found in the membrane, six are located near the ends of transmembrane segments close to the putative lipid/aqueous interface. This pattern is found in the experimental structures of helical membrane proteins deposited in the Protein Data Bank. As this bias was not used in the modeling, Trp location in the NIS secondary structure model was taken as an indication of the correctness of the helix assignments. Four Leu residues (positions 199, 206, 213, and 220) appear to comprise a putative leucine zipper motif in transmembrane segment VI. This motif could play a role in the possible oligomerization of subunits in the membrane. Indeed, subsequent freeze-fracture electron microscopy studies of X. laevis oocytes expressing NIS revealed the presence of 9-nm intramembrane particles corresponding to NIS (32). The size of these particles suggests that NIS may be an oligomeric protein. To date, five NIS hydrophilic segments (the NH₂ terminus, loops between transmembrane segments II and III, VI and VII, VIII and IX, and XII and XIII), of a total of seven, have been experimentally confirmed to have an external orientation (31), as predicted in the model (see Fig. 2 and Section II.C.2).

    fig.ommittedl, http://www.100md.com

    Figure 2. Current NIS secondary structure model. The model contains 13 putative transmembrane segments. The NH₂ terminus faces the extracellular milieu, and the COOH terminus faces the cytosol. Coordinates for the model were obtained with the program QUANTA (Molecular Simulations, Burlington, MA). Regularization of the model was carried out with the program O. Graphics were generated with the program SECTOR.l, http://www.100md.com

    The cDNA encoding hNIS was identified on the expectation that hNIS would be highly homologous to rNIS. Using primers to the cDNA rNIS sequence, Smanik et al. (33) identified a cDNA clone encoding hNIS. The nucleotide sequence of hNIS revealed an open reading frame of 1929 nucleotides, which encodes a protein of 643 amino acids. hNIS exhibits 84% identity and 93% similarity to rNIS. hNIS differs from rNIS mostly on account of a 5-amino-acid insertion between the last two hydrophobic domains and a 20-amino-acid insertion in the COOH terminus. Subsequently, Smanik et al. (34) examined the expression, exon-intron organization, and chromosome mapping of hNIS. Fifteen exons encoding hNIS were found to be interrupted by 14 introns, and the hNIS gene was mapped to chromosome 19p13. cDNAs encoding NIS have also been isolated from two other species, namely pig (35) and mouse (36). Mouse (36) and rNIS (3) contain 618 amino acid residues, whereas human (33) and pig NIS (35) contain 643. A very high sequence identity among all isolated NIS proteins exists (Figs. 3 and 4).

    fig.ommitted+-j, 百拇医药

    Figure 3. NIS cDNA and protein sequence identity in four species: human, pig, rat, and mouse. ORF, Open reading frame.+-j, 百拇医药

    fig.ommitted+-j, 百拇医药

    Figure 4. Alignment of amino acid sequences of human, pig, rat, and mouse NIS. The amino acid sequences of the human, pig, rat, and mouse NIS proteins were aligned based on the NIS consensus sequence (top). Dark areas indicate sequence divergence.+-j, 百拇医药

    B. NIS protein family+-j, 百拇医药

    NIS belongs to the sodium/solute symporter family [SSF, TC N° 2.A.21 (according to the Transporter Classification system; Ref. 37)] or solute carrier family 5 [SCL5A, according to the Online Mendelian Inheritance in Man (OMIM) classification, http://www3.ncbi.nlm.nih.gov/Omim/; Fig. 5]. This family includes more than 60 members of both prokaryotic and eukaryotic origin. All transport proteins in this family exhibit a high sequence similarity among them, and their functions are also remarkably close. Like NIS, all other members of the family rely on the Na⁺ electrochemical gradient as the driving force for solute transport into the cell. However, some important differences in cation selectivity and stoichiometry exist (see Section II.C.4).

    fig.ommitted5, 百拇医药

    Figure 5. Dendrogram of the sodium/solute symporter family. Percentage of identity was calculated using the Clustar method with PAM250 residue weight table. The scale shown at bottom left indicates relative phylogenetic distance.5, 百拇医药

    Studies carried out in several members of the family (30, 38, 39, 40) and computer predictions employing different methods, such as PredictProtein (http://www.embl-heidelberg.de/predictprotein; Ref. 41), suggest that virtually all members of the family share the 13-transmembrane-segment pattern (42) with the N terminus facing the extracellular milieu and the COOH terminus facing the cytosol, as described for NIS above (see Section II.B). In addition to NIS (30), this 13-transmembrane-segment pattern has also been proposed for the Na⁺/proline transporter (39). Other transporters in the family, such as the sodium/glucose cotransporter (SGLT) and the sodium/myo-inositol cotransporter (SMIT), have one additional transmembrane segment in the COOH terminus (38). It is precisely in the COOH terminus where sequence homology is the lowest among the various transporters.

    The eukaryotic members of the family include, besides NIS (SLC5A5), three different isoforms of the SGLT (SGLT1 or SLC5A1, SGLT2 or SLC5A2, and SGLT3), the SMIT (SMIT or SCL5A3), the sodium/proline symporter (NPT or PutP; Ref. 43), the sodium/multivitamin transporter (SMVT or SLC5A6; Refs. 44, 45), and the high-affinity choline transporter (46, 47). SMVT has the highest identity with NIS (35.9%). The sequence distance and rate of identity among these transporters are summarized in the phylogenetic tree (Fig. 5). The prokaryotic members of the family include the sodium-dependent transporters of proline (putP), pantothenate (panF), phenyl acetate (ppa), and glucose/galactose (vSGLT) (42, 48). Several additional sequences with as yet unknown functions have been predicted to belong to this family.cxu, 百拇医药

    C. The road to NIS characterizationcxu, 百拇医药

    1. N-linked glycosylation of NIS: implications for the NIS secondary structure model.cxu, 百拇医药

    The high-affinity anti-COOH terminus NIS Ab we generated (28) immunoreacts with a mature approximately 87-kDa polypeptide (i.e., NIS) and a partially glycosylated (~ 56 kDa) polypeptide in FRTL-5 cells. Immunoreactivity is also observed in X. laevis oocytes and COS cells expressing NIS and is competitively blocked by the presence of excess synthetic peptide. This anti-COOH terminus NIS Ab was the first available tool to experimentally probe the initial NIS secondary structure model; we used it to confirm the model-predicted cytosolic-side location of the carboxy terminus by indirect immunofluorescence experiments in permeabilized FRTL-5 cells (28). Our group has obtained conclusive evidence showing that neither partial nor total lack of N-linked glycosylation impairs activity, stability, or targeting of NIS (30). We demonstrated that, to a considerable extent, function, targeting, and stability of NIS are present even in the total absence of N-linked glycosylation (30). Therefore, a bacterial expression system, in which no N-linked glycosylation occurs, may be used to overproduce NIS for structural studies. In our report (30) of N-linked glycosylation of NIS, we demonstrated that the putative N-linked glycosylation site at N225, which had originally been predicted to face intracellularly, is indeed glycosylated. Therefore, this indicates that the hydrophilic loop that contains this sequence faces the extracellular milieu rather than the cytosol, as shown in the current 13-transmembrane-segment model (Fig. 2).

    2. Studies on NIS topology.%!/8q\, 百拇医药

    We have demonstrated unequivocally that the NH₂ terminus faces the external milieu, as proposed in the current model (30). This conclusion was reached using two independent experimental approaches. First, we introduced a FLAG (MDYKDDDDK) epitope into the NH₂ terminus. COS cells transfected with FLAG-containing NIS displayed indistinguishable I^- uptake accumulation from COS cells transfected with wild-type NIS. Immunofluorescence experiments demonstrated positive immunoreactivity with anti-FLAG Ab in nonpermeabilized COS cells transfected with FLAG-containing NIS. Positive immunoreactivity in nonpermeabilized cells indicates that the NH₂ terminus faces externally. In contrast, immunoreactivity using anti-COOH Ab requires permeabilization because the COOH terminus faces the cytosol. The second approach took advantage of the previous observation that unglycosylated NIS is active. The N-linked glycosylation amino acid sequence NNSS was introduced into the NH₂ terminus of unglycosylated NIS (31). We observed glycosylation of NIS at the NH₂ terminus upon transfection of NNSS-containing NIS into COS cells, thus proving that the NH₂ terminus faces the lumen of the endoplasmic reticulum during biosynthesis and therefore faces the external milieu upon reaching the plasma membrane (31).

    In addition, utilizing the same strategy of N-linked glycosylation scanning mutagenesis, we have demonstrated that the hydrophilic loop between putative transmembrane segments VIII and IX faces the external milieu (Fig. 2 and Ref. 31). In a complementary approach to study the topology of NIS in the plasma membrane, a Cys residue was placed at position 160 (in the hydrophilic loop between putative transmembrane segments IV and V) in an extracellular Cys-less background, a mutant that retains total activity. NIS activity was modified by membrane-impermeable sulfhydryl reagents such as sodium(2-sulfonatoethyl)metanethiosulfonate and 2-(trimethylamonium)ethyl methanethiosulfonate, indicating the external localization of this residue. In summary, as indicated earlier, five NIS loops (NH₂ terminus, loops between transmembrane segments IV and V, VI and VII, VIII and IX, and XII and XIII) of a total of seven have experimentally been confirmed to have the external disposition predicted in the current 13-transmembrane-segment secondary structure model.

    3. Structure/function studies of NIS. Findings derived from NIS mutations that cause congenital ITD.:1/q!!, 百拇医药

    We have demonstrated that a hydroxyl group at the ß-carbon at position 354 (in transmembrane segment IX) is essential for NIS function (49). Such a hydroxyl group is present in Thr 354. This discovery followed reports that a spontaneous mutation consisting of the single-amino-acid substitution of Pro instead of Thr at position 354 (T354P) is the cause of congenital lack of I^- transport in several patients (see Section VII and Ref. 50). Patients with this condition do not accumulate I^- in their thyroids, often resulting in severe hypothyroidism.:1/q!!, 百拇医药

    Significantly, transmembrane segment IX, in which Thr 354 is located, is where the highest incidence of hydroxyl-containing amino acids occurs in NIS. Hence, our group assessed the role played by these other hydroxyl groups in NIS function by replacing the corresponding amino acid residues with Ala and Pro (51). We observed that the hydroxyl groups of Ser 353, Ser 356, and Thr 357 seem to be essential for NIS activity, given that NIS functioned to a significant extent only when Ser or Thr was present at these positions. Interestingly, residues 353–357 face the same side of the helix on a helical wheel representation (Fig. 6).

    fig.ommittedf, 百拇医药

    Figure 6. -Helical wheel projection of the transmembrane segment IX of NIS. Hydroxyl-containing amino acid residues are circled. Important residues for NIS activity are shaded. The single-letter amino acid code is used. The inner numbers indicate position of the amino acid residues within the helix; the outer numbers indicate overall residue position in the NIS molecule.f, 百拇医药

    Using a similar approach, we analyzed G395R, another ITD-causing NIS mutation (52). We carried out a detailed study of the mechanism by which the G395R mutation renders NIS nonfunctional by analyzing G395R NIS protein processing, membrane targeting, and I^- transport in COS cells transiently transfected with G395R NIS cDNA. In addition, we probed the significance of the Gly395 residue by performing several additional amino acid substitutions at position 395 and evaluating the effects of these substitutions on the expression, plasma membrane targeting, and functional characteristics of the resulting NIS mutant constructs. We concluded that the presence of an uncharged amino acid residue with a small side-chain at position 395 is a requirement for NIS function, suggesting that glycine 395 is located in a tightly packed membrane-embedded region of NIS.

    4. Electrophysiological analysis of NIS: mechanism, stoichiometry, and specificity.dv, 百拇医药

    Our group, in collaboration with Ernest Wright’s group (32), has examined the mechanism, stoichiometry, and specificity of NIS by means of electrophysiological, tracer uptake, and electron microscopic methods in X. laevis oocytes expressing NIS. We obtained electrophysiological recordings using the two microelectrode voltage clamp technique and showed that an inward steady-state current (i.e., a net influx of positive charge) is generated in NIS-expressing oocytes upon addition of I^- to the bathing medium, leading to depolarization of the membrane. As the recorded current is attributable to NIS activity, this observation confirms that NIS activity is electrogenic. Simultaneous measurements of tracer fluxes and currents revealed that two Na⁺ ions are transported with one anion, demonstrating unequivocally a 2:1 Na⁺/I^- stoichiometry. Therefore, the observed inward steady-state current is due to a net influx of Na⁺ ions. In addition, we determined that the turnover rate of NIS at -50 mV is approximately 36 sec^-1 (Table 2) and reported that expression of NIS in oocytes led to an approximately 2.5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles, as ascertained by freeze-fracture electron microscopy. On the basis of our kinetic results, we proposed an ordered simultaneous transport mechanism in which Na⁺ binds to NIS before I^-, i.e., whereas transport of both ions is simultaneous, binding is ordered and sequential. Electrophysiological measurements and freeze-fracture electron microscopy suggest that NIS may be an oligomer.

    fig.ommitted}g6|, 百拇医药

    Table 2. Kinetic parameters of NIS in various systems}g6|, 百拇医药

    5. Effects of perchlorate (ClO₄^-) and thiocyanate (SCN^-) on NIS function.}g6|, 百拇医药

    It has long been known that certain large anions such as thiocyanate and perchlorate are competitive inhibitors of I^- accumulation in the thyroid (1, 17, 19). The antithyroid and goitrogenic properties of these anions were first discovered in 1936 when Barker et al. (53) reported occurrence of goiter and/or hypothyroidism as a side effect in patients treated with thiocyanate for hypertension. The mechanism of inhibition of perchlorate and thiocyanate involves a similarity in size and charge of the anions to I^-, so that the closer the ionic radius of the inhibitor to I^- the lower the K_i value (Table 2). This suggests that perchlorate and other inhibitors may interact with the same site as I^- on the NIS molecule, a property that was used to identify the I^- transport system in salivary glands, lactating mammary gland, stomach, choroid plexus, and fetal thyroid. Univalency is a requirement for inhibition, because divalent anions fail to inhibit transport (19). Thiocyanate is not concentrated in the thyroids of most species studied, because it is rapidly metabolized after translocation into the thyroid follicular cells (19). However, thiocyanate is concentrated by salivary tissue and gastric mucosa (19). Perchlorate (K_i = 1.7 µM) is 10–100 times more potent than thiocyanate as an inhibitor of I^- accumulation in a variety of in vivo and in vitro systems (Table 2). It was at one time also used in the treatment of hyperthyroidism but was eventually withdrawn in the United States because of severe adverse effects (aplastic anemia and agranulocytosis; Ref. 54). However, perchlorate is still used in some countries for the treatment of amiodarone-induced thyrotoxicosis (55). Table 2 shows the inhibition constants for perchlorate and thiocyanate along with NIS kinetic parameters reported in various systems.

    Perchlorate salts are found in rocket fuel, fireworks, and fertilizer. Perchlorate has recently been detected in the 4- to 18-µg/liter range in large public water supplies in several states in the United States (55), and this has caused concern at the Environmental Protection Agency (55, 56, 57). The daily ingestion of perchlorate at these levels would be considerably less than the doses that had been used in the treatment of hyperthyroidism, which ranged in the hundreds of milligrams. Still, a study by Lawrence et al. (57) clearly demonstrated the high sensitivity of thyroid NIS to perchlorate: a low dose of 10 mg/d during 14 d given to human volunteers significantly decreased thyroid radioiodide accumulation without affecting the levels of circulating thyroid hormones or TSH.*|, http://www.100md.com

    Both thiocyanate and perchlorate have been shown to cause the rapid discharge of accumulated I^- from PTU-blocked thyroid tissue across the basolateral membrane toward the interstitium (58, 59). This phenomenon is the basis for the perchlorate discharge test, the purpose of which is to detect defects in intrathyroidal I^- organification. In normal subjects, the administration of perchlorate blocks the continued accumulation of radioiodide by the thyroid but causes virtually no release of previously accumulated radioiodide from the gland. In contrast, in patients with an I^- organification defect, administration of the inhibitor results in the release of I^- from the thyroid. The efficacy of I^- organification needs to be evaluated in certain pathological conditions, such as organification genetic defects (60).

    It was believed for a long time that perchlorate was translocated via NIS into the thyroid follicular cells (19, 56). However, we have reported that whereas I^- and a wide variety of other anions (including ClO₃^-, SCN^-, SeCN^-, NO₃^-, Br^-, BF₄^-, IO₄^-, and BrO₃^-) generated steady-state inward electrical currents in X. laevis oocytes expressing rNIS, perchlorate did not (Table 2, Fig. 7, and Ref. 32). This suggested that perchlorate was not translocated into the oocytes, although electroneutral transport could not be excluded. Earlier experiments ostensibly showing that ³⁶Cl-perchlorate enters the cell were probably misinterpreted. ³⁶Cl-Chlorate (ClO₃^-), rather than perchlorate, accounted for the presence of radioactivity in the cytosol of thyrocytes, given that chlorate is readily translocated via NIS into the cell. ³⁶Cl-Chlorate is a ³⁶Cl byproduct of the reaction employed to chemically synthesize ³⁶Cl-perchlorate. Yoshida and colleagues (61, 62) have also reported that perchlorate did not induce an inward current in FRTL-5 cells (61) or in Chinese hamster ovary (CHO) cells stably expressing NIS (62). Hence, it is clear that perchlorate is not translocated via NIS into the cell and that it acts as a blocker rather than a substrate.

    fig.ommitted;k, 百拇医药

    Figure 7. Substrate selectivity of NIS. Inward currents induced by various anions (500 µM) were recorded at V_m = -50 mV. Currents were normalized with respect to the current generated by I^-. I^--induced current in the absence of Cl^- did not differ from that in the presence of 100 mM Cl^-. Other anions tested, which did not induce a detectable inward current, were: NO₂^-, HCO₃^-, SO₃^2-, CO₃^2-, S₂O₃^2-, and HPO₄^2-. Data are reported as mean ± SE (n = 3). Adapted from Ref. 32 .;k, 百拇医药

    III. Transcriptional Regulation of NIS;k, 百拇医药

    It has long been established that TSH stimulates NIS activity via the cAMP pathway (28, 29, 63) and, more recently, that it up-regulates NIS mRNA levels (64). However, to fully understand these mechanisms it is necessary to study the transcriptional regulation of NIS. The rNIS and hNIS promoters have been studied by several groups (65, 66, 67, 68, 69, 70, 71, 72, 73, 74). These findings have been extensively reviewed elsewhere (7, 8, 9, 10, 11, 12, 13, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74). Figure 8 is a schematic representation of the rNIS promoter structure.

    fig.ommitted?-7|f, 百拇医药

    Figure 8. Schematic representation of the rNIS promoter structure. Diagram of the NIS promoter indicating the major transcription start site (+1), the TATA box (AATAAAT), the proximal promoter, and the NIS upstream enhancer (NUE). The proximal promoter contains a thyroid transcription factor 1 (TTF1) binding site and a TSH responsive element where a putative transcription factor NTF-1 (NIS TSH-responsive factor-1) interacts. NUE contains two Pax8 binding sites and a degenerate CRE (cAMP responsive element sequence), which are important for full TSH-cAMP-dependent transcription.?-7|f, 百拇医药

    IV. Regulation of NIS Expression and Function?-7|f, 百拇医药

    A. TSH?-7|f, 百拇医药

    TSH is an approximately 30-kDa glycoprotein biosynthesized in the adenohypophysis by basophilic cells known as thyrotropes. TSH is the primary hormonal regulator of thyroid function overall and stimulates I^- accumulation in the thyroid (75). TRH from the hypothalamus stimulates the release of TSH, whereas T₃ and T₄ inhibit it. The majority of TSH actions are mediated by activation of adenylate cyclase via the GTP binding protein G{alpha} s (76). This series of events starts with the interaction of TSH with the TSH receptor (TSHR) on the basolateral membrane of the follicular cells (Fig. 1). TSH stimulation of I^- accumulation was known to result from the cAMP-mediated increased biosynthesis of NIS (63). Using high-affinity anti-NIS Abs, we demonstrated in rats that NIS protein expression is up-regulated by TSH in vivo (28). Consistent with these findings is a later observation by Uyttersprot et al. (77) that the expression of NIS mRNA in dog thyroid (~ 3.9 kb) is dramatically up-regulated by goitrogenic treatment (i.e., PTU treatment, which leads to elevated TSH circulating levels in vivo). Moreover, no thyroidal I^- uptake is detected in humans whose serum TSH levels are suppressed (78).

    Up-regulation of thyroid NIS expression and I^- uptake activity by TSH has been demonstrated not only in rats in vivo (28) but also in the rat thyroid-derived FRTL-5 cell line (63) and in human thyroid primary cultures (79, 80). Marcocci et al. (81), Kogai et al. (64), and Ohno et al. (71) have all shown that TSH up-regulates I^- uptake activity by a cAMP-mediated increase in NIS transcription. After TSH withdrawal, a reduction of both intracellular cAMP levels and I^- uptake activity is observed in FRTL-5 cells (63). This is a reversible process, as I^- uptake activity can be restored either by TSH or agents that increase cAMP (63, 71). To investigate NIS biogenesis, our group carried out metabolic labeling and immunoprecipitation experiments in the presence of TSH and observed that NIS is synthesized as a precursor of approximately 56 kDa (82). After a 60-min chase period, a broad approximately 87-kDa polypeptide band also became apparent, whereas the intensity of the approximately 56-kDa band decreased. The approximately 56-kDa precursor disappeared by 180 min, at which time only the approximately 87-kDa band, presumably fully processed NIS, was visible.

    We made the surprising observation that I^- uptake activity persists in membrane vesicles (MV) prepared from FRTL-5 cells that, when intact, have completely lost I^- uptake activity due to prolonged TSH deprivation (83). This suggested that mechanisms other than transcriptional ones might also operate to regulate NIS activity in response to TSH. Our group has more recently demonstrated conclusively by immunoblot analysis that NIS is present in FRTL-5 cells as late as 10 d after TSH withdrawal and that de novo NIS biosynthesis requires TSH (82). Therefore, it is clear that any NIS molecules detected in TSH (-) FRTL-5 cells had to be synthesized before TSH withdrawal. This is consistent with NIS being a protein with an exceptionally long half-life, as previously suggested by Kogai et al. (64) and Paire et al. (29). We determined by pulse-chase analysis that the NIS half-life is approximately 5 d in the presence and approximately 3 d in the absence of TSH. Even though the NIS half-life in the absence of TSH is 40% shorter than that in the presence of the hormone, it is still sufficiently long to account for the persistence of significant I^- uptake activity in MV from cells deprived of TSH (82).

    Kogai et al. (79) have shown that TSH markedly stimulates NIS mRNA and protein levels in both monolayer and follicle-forming human primary culture thyrocytes, whereas significant stimulation of I^- uptake is observed only in follicles. These interesting observations indicate that, in addition to TSH stimulation, cell polarization and spatial organization are also crucial for proper NIS activity and suggest that NIS may be regulated by such posttranscriptional events as subcellular distribution. Indeed, we later observed that 3 d after TSH deprivation, intracellular NIS decreases at a slower rate than plasma membrane NIS, supporting the notion that active NIS molecules, initially located in the plasma membrane while TSH is present, are redistributed to intracellular compartments in response to TSH withdrawal (82). This model (Fig. 9) explains the presence of NIS activity in MV from cells deprived of TSH that, when intact, exhibit no NIS activity. Clearly, TSH regulates I^- uptake by modulating the subcellular distribution of NIS without apparently influencing the intrinsic functional status of the NIS molecules. In conclusion, TSH not only stimulates NIS transcription and biosynthesis, it is also required for targeting NIS to and/or retaining it at the plasma membrane.

    fig.ommitted0'', 百拇医药

    Figure 9. TSH effects on NIS expression and function in thyroid cells. A, I^- transport activity. FRTL-5 cells were kept in the presence of TSH or in its absence for 5 d. I^- transport activity was measured in intact cells (green bars) and in MV prepared from these cells (red bars). I^- transport activity was expressed as the percentage of I^- transport relative to that observed in the presence of TSH (82 ). B, NIS protein expression in FRTL-5 cells kept in the presence or 5 d after withdrawal of TSH. MV from these cells were prepared, electrophoresed, electrotransferred, and analyzed by immunoblot using a high-affinity anti-NIS Ab (82 ). C, Schematic model to illustrate the presence of NIS activity in MV from cells deprived of TSH that, when intact, exhibit no NIS activity. This model supports the notion that active NIS molecules, initially located at the plasma membrane while TSH is present, are redistributed to intracellular compartments in response to TSH withdrawal. NIS molecules are represented as cylinders. The plasma membrane is shown in red, ER and Golgi in blue, and intracellular compartments in green. D, NIS immunofluorescence in FRTL-5 cells as analyzed by confocal microscopy using an anti-NIS Ab. Upper panel, FRTL-5 cells kept in the presence of TSH; lower panel, FRTL-5 cells 5 d after withdrawal of TSH (82 ).

    The molecular mechanisms by which NIS is targeted to, retained at, and retrieved from the basolateral plasma membrane are unknown. NIS contains several sorting signals in its COOH terminus that, in other membrane proteins, are involved in the targeting, retention to, and endocytosis from the plasma membrane (84, 85, 86). NIS contains a PDZ target motif (T/S-X-V/L) at the COOH-terminal tail (T⁶¹⁶N⁶¹⁷L⁶¹⁸), a motif that is one of the sequences involved in protein-protein interactions. PDZ target sequences are recognized by PDZ binding proteins (84). Among the various proteins that contain PDZ domains (84) is PDZ binding protein LIN-7, which, by recognizing a PDZ target motif in the epithelial {gamma} -aminobutyric transporter, prevents its internalization from the basolateral surface of polarized epithelial cells (87).\[i|+, http://www.100md.com

    NIS also contains a dileucine motif, L⁵⁵⁷L⁵⁵⁸, which has been proposed to play a role in the sorting of certain membrane proteins within the cell (85, 88). The dileucine motif, like tyrosine-based sorting signals, interacts directly with the clathrin-coated machinery (89). This interaction allows for selective incorporation of the integral membrane proteins into coated vesicles that carry proteins to different destinations within the cell. In addition, three acidic dipeptide motifs are present in the COOH terminus of NIS, namely E⁵⁷³D⁵⁷⁴, E⁵⁷⁹E⁵⁸⁰, and E⁵⁸⁷D⁵⁸⁸. Acidic-based motifs function as retrieval signals for proteins localized at the cell surface (86, 90). These signals also function as retention signals in large dense core vesicles, as in the case of the vesicular monoamine transporter (91).

    Localization of NIS at the basolateral plasma membrane is not only important for I^- transport in the thyroid gland, it is also essential for radioiodide therapy in thyroid cancer (see Section IX). The decrease in I^- uptake observed in most thyroid cancers is due to impaired NIS targeting to or retention at the plasma membrane (92, 93). Therefore, it is of considerable interest to elucidate the mechanisms that regulate the subcellular distribution of NIS.|h\qn2/, http://www.100md.com

    B. Posttranscriptional regulation of NIS|h\qn2/, http://www.100md.com

    Phosphorylation, a common cellular mechanism for modulating activity, subcellular localization, and/or degradation of proteins, has recently been reported to play a role as a posttranscriptional regulatory mechanism for the activity of transporters (94, 95, 96, 97, 98, 99). NIS contains several consensus sites for kinases, including glycogen synthase kinase 3, cyclin-dependent kinases I and II, protein kinase A (PKA), and protein kinase C. We have shown that NIS is phosphorylated in vivo and that serines are the main amino acid residues in which phosphorylation takes place in NIS, independently of TSH presence (82, 100). However, the phosphopeptide map of NIS obtained when TSH was present was markedly different from that when TSH was absent (82). Five phosphopeptides were resolved in the presence and three in the absence of TSH (82). Only one among these phosphopeptides seemed to be common to both conditions, as calculated by the migration coefficient (82). As TSH actions in the thyroid are mainly mediated by cAMP and given that phosphorylation has been reported to play a role in regulating the targeting of other transporters, it is possible that phosphorylation might be involved in the regulation of NIS subcellular distribution.

    C. Regulation of NIS activity by I^-6j][((, http://www.100md.com

    1. Recent research on the Wolff-Chaikoff effect.6j][((, http://www.100md.com

    The main factor regulating the accumulation of I^- in the thyroid (i.e., NIS activity), other than TSH, has long been considered to be I^- itself. Stated simply, high doses of I^- cause diminished thyroid function. Plummer (101), in 1923, was the first to administer high doses of I^- to block thyroid function. In 1944, Morton et al. (102) reported that the biosynthesis of thyroid hormones by sheep thyroid slices was inhibited by high doses of I^-. Wolff and Chaikoff (103) reported in 1948 that organic binding of I^- (i.e., I^- organification, which years later was determined to be mediated by TPO) in the rat thyroid in vivo was blocked when I^- plasma levels reached a critical high threshold, a phenomenon known as the acute Wolff-Chaikoff effect. I^- organification resumed when I^- plasma levels fell. Wolff and Chaikoff concluded that this effect could be the mechanism by which administration of high I^- doses results in remission of Graves’ disease. Raben (104) observed that blocking I^- transport with thiocyanate prevented the inhibiting effect of high plasma I^- levels, concluding that acute inhibition of organic I^- binding depends on the intrathyroidal rather than the plasma concentration of I^-. Despite having been extensively investigated by several groups over the years, the precise mechanism underlying the inhibition of I^- organification by high levels of I^- remains poorly understood. I^- was subsequently found to inhibit the TSH-induced increase of cAMP formation in vitro in dog thyroid slices, but this inhibitory effect disappeared when MMI (a TPO inhibitor) was given together with I^- (104).

    Grollman et al. (105) observed that I^- preincubation suppressed I^- uptake activity in FRTL-5 cells in a time- and dose-dependent manner. Interestingly, the presence of MMI during the incubation period abolished the I^- uptake-suppressing effect of I^- (105). This action of MMI, observed both in vivo and in vitro, suggested that the Wolff-Chaikoff effect of I^- is mediated by an intracellular iodinated compound. The proposed candidates were iodolipids (6-iodo-5-hydroxy-8,11,14-eicosa-trienoic acid {delta} -lactone and {alpha} -iodohexadecanal) that can be formed from arachidonic acid in the presence of H₂O₂ (106). These compounds have been shown to inhibit the TSH-stimulated adenylate cyclase activity (107, 108).dza$], http://www.100md.com

    Wolff et al. (109) reported in 1949 that the maximum duration of the inhibitory effect of high concentrations of I^- on I^- organification was 50 h in the presence of continued high plasma I^- concentrations. However, as early as 2 d after onset of the acute effect, an escape or adaptation from the effect occurred, so that the level of organification of I^- was restored and normal hormone biosynthesis resumed. In 1963, Braverman and Ingbar (110) investigated in detail in rats the mechanism underlying the escape from the acute Wolff-Chaikoff effect. These authors studied in vitro the I^- uptake capability of the thyroid after the gland adapted in vivo to high I^- levels, as compared with control nonadapted thyroids. They found that the adapted glands concentrated far less I^- than control glands. In addition, they observed that inhibition of I^- organification by high external I^- concentrations in vitro was much more pronounced in the nonadapted than the adapted glands. On this basis, Braverman and Ingbar proposed that the escape from the acute Wolff-Chaikoff effect was due to a decrease in I^- transport, which would presumably lead to sufficiently low intracellular I^- concentrations to remove inhibition of I^- organification. The Wolff-Chaikoff effect and the ensuing escape constitute a highly specialized intrinsic autoregulatory system that protects the thyroid from the deleterious effects of I^- overload but at the same time ensures adequate I^- uptake for hormone biosynthesis. The level of I^- capable of inhibiting I^- organification and concomitantly stopping thyroid hormone synthesis is determined by the ratio of organified to nonorganified intracellular I^- content, which in turn depends on the previous I^- supply status of the animal.

    As in the case of NIS regulation by TSH, the regulatory role played by I^- on NIS function began to be explored at the molecular level only after the cDNA that encodes NIS was isolated. In fact, isolation of the cDNA that encodes NIS has spurred a renewed impetus to investigate this topic. In vivo studies carried out by Uyttersprot et al. (77) showed that I^- inhibited the expression of both TPO and NIS mRNAs in dog thyroid, although NIS protein levels were not measured. These observations support the proposed mechanism to explain the escape from the Wolff-Chaikoff effect, i.e., that it is due to a decrease in I^- uptake possibly caused by down-regulation of NIS expression./a7yfry, 百拇医药

    Spitzweg et al. (111) investigated the effects of I^- and several other agents on I^- transport activity, NIS mRNA, and NIS protein levels in FRTL-5 cells by I^- uptake assays and Northern and Western blot analysis. They reported a 50% decrease in both I^- uptake and NIS mRNA levels. However, the authors did not carry out immunoblot analysis of NIS expression and did not discuss the possibility that preincubation with I^- might have resulted in higher intracellular I^- concentrations, thus complicating interpretation of the results. In 1999, Eng et al. (112) reinvestigated at the molecular level their earlier hypothesis on the escape from the acute Wolff-Chaikoff effect. They found that both NIS mRNA and NIS protein levels decreased significantly after either 1 or 6 d of I^- administration. NIS mRNA levels were already significantly reduced at 6 h following the injected single dose of I^-. In contrast, a significant decrease of NIS protein levels was detected only at 24 h. These findings were not correlated with NIS activity by thyroid scintigraphy. The conclusion of this study was that the decrease in active I^- transport, i.e., the basis for the escape, occurs between 6 and 24 h by a mechanism that at least in part involves a decrease in NIS transcription.

    Eng et al. (113) later investigated the effect of I^- on NIS mRNA and protein expression in FRTL-5 cells. Incubation of FRTL-5 cells with I^- (10^-3 M) did not affect NIS mRNA levels, but NIS protein levels decreased significantly in a dose-dependent manner. This conflicts with the authors’ previous in vivo observations (112) and with the findings of Spitzweg et al. (111), who reported a 50% decrease in NIS mRNA levels in FRTL-5 cells incubated with I^- (10^-4 M). When I^- was administered during TSH stimulation (72 h after TSH deprivation), the increase in NIS protein levels was less pronounced in the I^--treated cells than in the controls. Performing pulse-chase experiments, the authors found that the half-life of the NIS protein was shorter in the I^--treated cells, suggesting increased NIS protein turnover in these cells. However, the half-life of NIS reported in this study in normal nontreated FRTL-5 cells was less than 24 h, which is much shorter than the 4–5 d reported by several other groups (28, 82, 29, 64). In summary, the authors concluded that high doses of I^- administered in vivo lead to decreases in both NIS mRNA and protein levels by a mechanism that is likely to be at least in part transcriptional, whereas their studies in vitro suggested that the I^--induced decrease in NIS protein levels appears to be due at least in part to an increase in NIS protein turnover.

    NIS regulation is fairly complex. Whereas the NIS protein is distributed both in the plasma membrane and in intracellular membrane compartments, NIS activity derives only from NIS protein molecules located in the plasma membrane (82, 83). The subcellular distribution of NIS is regulated mainly by TSH (82, 83). Hence, further investigation is necessary to examine the parallel assessment of NIS mRNA and protein expression, cellular distribution, and function, both in vitro and in vivo, to better understand the regulatory effects exerted by I^- on NIS.8-l$g, 百拇医药

    2. Stunning.8-l$g, 百拇医药

    Radioiodide is the cornerstone of the treatment of metastatic thyroid cancer. The optimal therapeutic radioiodide dose is calculated on the basis of the scintigraphic image obtained upon administration of a radioiodide test dose. This test dose must be properly adjusted so as to prevent uptake inhibition of the subsequently administered therapeutic dose of ¹³¹I^-. The interference of radioiodide test doses with uptake of subsequent therapeutic doses is called stunning, the molecular mechanism of which is unknown.

    To investigate stunning, TSH-prestimulated primary cultures of pig thyrocytes [grown in a bicameral chamber, where vectorial (basal to apical) I^- transport can be assessed] were exposed to increasing doses of ¹³¹I^- or ¹²³I^- (1–100 Gy, iodide < 10^-9 M) for 48 h in the presence of TSH and MMI. Basal to apical I^- transport was then measured using ¹²⁵I^- (114). Immediately after exposure to radioiodide, active I^- transport was similar to the control. However, 3 d after ¹³¹I^- or ¹²³I^- exposure, basal to apical I^- transport decreased in a radioiodide dose-dependent manner. The presence of perchlorate or lack of TSH during initial radioiodide exposure prevented subsequent stunning. Based on these observations, the authors concluded that stunning of I^- accumulation after radioiodide exposure is due to selective inhibition of the I^- transporting mechanism.

    D. Effect of cytokines on NIS7\8eh, http://www.100md.com

    In addition to TSH and I^-, cytokines have also been shown to play a role in the modulation of NIS function in thyroid cells. Cytokines that affect thyroid function and growth and cause immunological changes in the gland are produced by both infiltrating inflammatory cells and the thyroid follicular cells themselves, albeit the latter only in autoimmune thyroid disease (115). The thyroidal effects of cytokines have mostly been examined in FRTL-5 cells kept in TSH-free medium, to which TSH and cytokines were then added simultaneously (111). The cytokines investigated include TNF-{alpha} , TNF-ß, interferon-{gamma} (IFN-{gamma} ), IL-1{alpha} , IL-1ß, IL-6, and TGF-ß₁, all of which exerted an inhibitory effect on thyroid function, including decreased NIS expression and I^- uptake.7\8eh, http://www.100md.com

    Ajjan et al. (115) and Spitzweg et al. (111) have reported that, in FRTL-5 cells, TNF{alpha} inhibited TSH-stimulated NIS mRNA expression and I^- uptake. NIS expression was studied by semiquantitative RT-PCR and Southern blot analysis (115) as well as by Northern blot analysis (111). In addition, Pekary et al. (116) reported that activation of sphingomyelinase—an enzyme that converts sphingomyelin to ceramide—by TNF led to inhibition of NIS expression. TNF reduced the activity and mRNA levels of the Na⁺/K⁺ ATPase and inhibited the conversion of T₄ to T₃ by type I deiodinase (117). The effects of TNF{alpha} and -ß were also studied in human thyroid cells in culture, in which a dose-dependent decrease of cAMP levels and Tg expression was observed (118). This effect was enhanced when TNFs were added together with IL-1ß (111).

    TGF-ß had a similar effect to TNF, i.e., it also inhibited I^- uptake and NIS mRNA in a time- and dose-dependent manner. TGF-ß similarly reduced the activity and mRNA levels of the Na⁺/K⁺ ATPase in a time- and dose-dependent manner in young FRTL-5 cells. However, in contrast to TNF, TGF-ß induced a change in young FRTL-5 cells from a cuboidal to a flattened stellate morphology. As FRTL-5 cells aged, an increase in TGF-ß expression and secretion was observed, which in turn reduced both NIS mRNA levels and I^- transport (116, 117). Contradictory results have been obtained when studying the effects of IFN- on NIS in FRTL-5 cells. Whereas Spitzweg et al. (111) reported that IFN- had no effect on I^- accumulation or NIS mRNA, Ajjan et al. (115) observed that IFN- at a high concentration (1000 U/ml) down-regulated TSH-stimulated NIS mRNA levels. IFN- at concentrations of 100 and 1000 (but not at 10) U/ml inhibited I^- transport. IFN- inhibited cAMP production and Tg expression in human thyroid cells in culture and, when combined with TSH, IFN- inhibited the TSH-stimulated functions of human thyroid epithelial cells. IFN-{gamma} was also tested in conjunction with IL-1ß, which showed that at low concentrations of IFN-, cAMP generation was stimulated with no effect on Tg expression. At high concentrations of IFN-, Tg levels were decreased by the enhanced effect of IL-1ß (118). Confirming and extending the observations of Ajjan et al., Caturegli et al. (119) reported the effect of IFN-{gamma} on thyroid function in vivo in transgenic mice expressing IFN- in the thyroid. IFN-{gamma} caused significant growth retardation, reduced fertility, severe impairment of thyroid function, loss of typical follicular structure, and suppressed NIS gene transcription, NIS protein expression, and I^- uptake activity.

    Interleukins exert effects similar to those of TNFs, TGF-ß, and IFN- on NIS regulation. High concentrations of IL-1 inhibited and low concentrations stimulated human thyroid cell function in vitro. IL-1{alpha} at concentrations of 100 and 1000 U/ml inhibited both basal and TSH-induced NIS expression in a dose-dependent manner, as well as I^- uptake. Spitzweg et al. (111) observed that IL-6, IL-1{alpha} , and IL-1ß caused a decrease in NIS mRNA and I^- uptake in all cases but to varying degrees. IL-1 had inhibitory effects on cAMP production and Tg levels, as was the case with the other cytokines (118). In conclusion, the interleukins tested caused decreases in NIS mRNA levels and I^- uptake activity in young cells. In aged cells, cytokines led to only a modest reduction in NIS mRNA levels, an effect that was not enhanced by addition of other cytokines (116, 117). Further studies are needed to elucidate the changes that FRTL-5 cells undergo with age.

    E. Tg+, http://www.100md.com

    As discussed earlier, NIS activity is up-regulated by TSH. Kohn’s group (120) has reported the intriguing observation that Tg acts as a potent suppressor of NIS mRNA levels and thyroid-restricted genes (i.e., Tg, TPO, and TSHR) in FRTL-5 cells and suggested that Tg could counterbalance the effect of TSH on these genes. The notion of Tg acting as a NIS suppressor is surprising because of the characteristics of the Tg molecule. Tg is synthesized as a 12S molecule that forms a 19S dimer and a 27S tetramer (121, 122). Using 19S follicular Tg (at concentrations known to exist in the follicular lumen) Kohn et al. (123) reported that follicular Tg suppressed TSH-increased NIS activity in vitro and in vivo and regulated the Tg, TPO, and TSHR genes at the transcriptional level (123). Purified 12S, 19S, and 27S follicular Tg suppression of thyroid-restricted gene expression was dependent on their ability to bind to FRTL-5 thyrocytes (124). This binding was blocked by an Ab against the thyroid apical membrane asialoglycoprotein receptor, which is a phosphoprotein that is critical for ATP-mediated inactivation of receptor-mediated endocytosis (124).

    F. Estradiolg.e}2wj, http://www.100md.com

    It has been proposed that the increased amount of estrogen in women may contribute to their increased susceptibility to goiter (125). Indirect effects of estradiol on thyroid function include an increase in T₄-binding globulin. An increase in cell growth and the reduced expression of the NIS gene are two direct effects of estradiol on thyroid follicular cells. In previous studies, Furlanetto et al. (125) reported that estrogen receptors are present in FRTL-5 cells and that a range of estrogen concentrations (between 10^-11 and 10^-7 M) caused an increase in cell growth (in the presence and absence of TSH) and reduced NIS expression. In later studies, using FRTL-5 cells as a model, Furlanetto et al. (126) reported that estradiol decreased I^- uptake in the presence and absence of TSH. Goiter formation may be promoted by the increase in cell growth and the reduction of NIS gene expression caused by estrogen, which would explain the higher prevalence of goiter in women compared with men.

    V. Signal Transductionr;i-, 百拇医药

    Hormones and growth factors exert their effects on thyroid cells via several signal transduction pathways. The TSH-TSHR-cAMP-PKA pathway has for a long time been considered the central and most important pathway for thyroid cell proliferation and differentiation (127). This is in contrast to many other cell types, in which cAMP inhibits growth (128, 129, 130). This pathway has been reported to play a role in the regulation of NIS expression, one of the markers of thyroid cell differentiation (71). Other markers include Tg and TPO (131, 132, 133). Interestingly, recent evidence indicates that cAMP pathways, both PKA dependent and independent, contribute to thyroidal cell differentiation and therefore NIS expression (134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151). The coexistence of PKA-dependent and -independent pathways for thyroid cell proliferation is not incidental and contributes to the establishment of the overall balance between these two complementary pathways. By itself, each pathway is insufficient to induce mitogenesis in thyroid cells (137, 148). As the information on TSH-dependent signal transduction pathways is becoming increasingly complex, the currently available data will likely turn out to be just a partial picture of the multiple interactions necessary to maintain the mitogenic capacity of thyroid cells without impairing their differentiated state.

    VI. Extrathyroidal NIS Expression5, 百拇医药

    The field of I^- transport systems outside the thyroid has changed considerably since the extensive review published on the topic in 1961 by Brown-Grant (152). The main vertebrate nonthyroid tissues reported to actively accumulate I^- are salivary glands, gastric mucosa, lactating mammary gland, choroid plexus, and the ciliary body of the eye. Many of these transport systems exhibit functional similarities with their thyroid counterpart, notably a susceptibility to inhibition by thiocyanate and perchlorate. However, they also display important differences: 1) nonthyroid I^- transporting tissues do not have the ability to organify accumulated I^- (with the possible exception of the lactating mammary gland); therefore, they behave like PTU-treated thyroid tissue; 2) TSH exerts no regulatory influence on nonthyroid I^- accumulation; 3) at least salivary glands and gastric mucosa concentrate thiocyanate, unlike the thyroid, in which thiocyanate is metabolized after uptake and therefore not concentrated. Despite these differences, several reports of patients suffering the simultaneous genetic absence of I^- transport in the thyroid, the salivary glands, and the gastric mucosa strongly hinted at a genetic link among these I^- transport systems, suggesting that extrathyroidal I^- transport is catalyzed by plasma membrane proteins that are very similar, if not identical, to thyroid NIS (1, 18, 103, 152). Moreover, thyroidal and extrathyroidal I^- concentration gradients are of similar magnitude (~ 20- to 40-fold under steady-state conditions). Hence, the isolation and characterization of the NIS cDNA from rat thyroid (3) and the generation of anti-NIS Abs (28) have made it possible to examine NIS expression in nonthyroid tissues, leading to the conclusion that I^- transport in most (and probably all) extrathyroidal tissues in which it is present is also mediated by NIS, as in the thyroid. However, NIS is clearly regulated and processed differently in each tissue.

    The cloning of hNIS cDNAs has been reported from gastric mucosa and parotid and mammary glands, all of which exhibited full identity to thyroid hNIS cDNA. Whereas hNIS gene expression has been detected in many other tissues by RT-PCR (Table 3 and Refs. 34, 36, 153, 154, 155, 160, 163), it must be pointed out that the RT-PCR technique yields a large number of false positives due to its high sensitivity (165). Therefore, the detection of the NIS-amplified product by RT-PCR in a given tissue cannot be regarded as sufficient evidence that NIS is functionally expressed in that tissue. A thorough characterization of NIS protein expression is necessary to properly evaluate the significance of results obtained by RT-PCR and Northern analysis. Still, as shown in Table 3, even with the use of a wide variety of techniques (Northern analysis, RT-PCR, Western analysis, and immunohistochemistry), different groups have often obtained inconsistent and sometimes conflicting results on whether NIS is expressed in a particular tissue. Hence, once NIS protein expression has been demonstrated, a correlation with Na⁺-dependent, perchlorate-sensitive, active I^- accumulation in that tissue must be established. By these criteria, and taking into consideration the above results, NIS is expressed and active in extrathyroidal tissues previously known to exhibit NIS activity, such as salivary glands, gastric mucosa, and lactating mammary gland (Fig. 10). The significance of the detection of the RT-PCR-amplified NIS product in other human and rat tissues remains to be ascertained.

    fig.ommitted-nxxw@k, http://www.100md.com

    Table 3. NIS expression and activity in different tissues-nxxw@k, http://www.100md.com

    fig.ommitted-nxxw@k, http://www.100md.com

    Figure 10. Immunoblot of healthy and diseased NIS-expressing human tissues. Membrane fractions from all tissues were prepared as described, electrophoresed on a 9% sodium dodecyl sulfate-polyacrylamide gel, and electrotransferred onto nitrocellulose (14 ). The nitrocellulose was incubated with 0.5 µg/ml of affinity-purified anti-hNIS Ab, followed by 0.3 µg/ml horseradish peroxidase-labeled goat antirabbit Ab (Amersham Biosciences). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham). Lane 1, Mammary gland from a pregnant woman (44 µg); 2, breast adenocarcinoma (100 µg); 3, mixed tumor of the salivary gland (20 µg); 4, gastric mucosa (20 µg); 5, multinodular goiter (20 µg); 6, thyroid follicular adenoma (20 µg); 7, thyroid papillary carcinoma (follicular variant, 20 µg); 8, thyroid from a patient with Graves’ disease (8 µg); 9, hNIS stably transfected Madin-Darby kidney epithelial cells (MDCK) (5 µg).

    A. Mammary gland NIS (mg-NIS)a9swzq., http://www.100md.com

    Physiologically, I^- transport in the mammary gland occurs during late pregnancy and lactation. An adequate supply of I^- for sufficient thyroid hormone production is essential for proper development of the newborn’s nervous system, skeletal muscle, and lungs. Our group (14) performed immunoblot analyses to assess whether a high-affinity anti-NIS Ab would react with a mammary gland membrane protein. We observed immunoreactivity against a single, broad, approximately 75-kDa polypeptide in rat lactating mammary gland membranes but not in membranes from nonlactating mammary gland or from lung, muscle, or heart, all tissues that do not transport I^-. This immunoreactive polypeptide is mg-NIS. We then investigated the difference in electrophoretic mobilities between mg-NIS (~ 75 kDa) and thyroid NIS (~ 90 kDa) and found that it is due to differences in their posttranslational modifications. We treated membrane proteins from thyroid and lactating mammary gland with N-glycosidase F, an enzyme that removes N-linked carbohydrates, and probed membranes with anti-NIS Ab. Under these conditions, anti-NIS Ab recognized an approximately 50-kDa polypeptide in membranes from both thyroid and lactating mammary gland. Significantly, both nonglycosylated NIS in FRTL-5 cells and NIS expressed in Escherichia coli exhibit an identical electrophoretic mobility (i.e., ~ 50 kDa). These results demonstrate that the approximately 75-kDa and approximately 50-kDa immunoreactive polypeptides detected in lactating mammary gland correspond to glycosylated and nonglycosylated mg-NIS, respectively. Cyanogen bromide treatment of rat thyroid NIS and mg-NIS proteins yielded the same peptide map (14), a finding consistent with the identity between human thyroid NIS and mg-NIS predicted by the cloning of hNIS cDNAs from mammary glands by Spitzweg et al. (154).

    mg-NIS hormonal regulation has been studied in vitro and in vivo. Rillemma et al. (166) showed that PRL stimulates I^- uptake in mammary gland explants. We observed that NIS is absent in mammary glands from nubile rats and that NIS expression was increasingly detectable toward the end of gestation and intensely apparent in lactating mammary gland (14). Interestingly, NIS expression was regulated in a reversible manner by suckling during lactation. In vivo studies in ovariectomized mice showed that the combination of ß-estradiol, oxytocin, and PRL led to the highest level of NIS expression.or\ot), 百拇医药

    B. NIS in the gastrointestinal tractor\ot), 百拇医药

    As indicated above, the functional role of NIS in salivary glands and in gastric and rectal mucosa is unknown. In the salivary glands, NIS protein has been detected in the basolateral membrane of all ductal epithelial cells (see Fig. 14; Refs. 14, 157, 158). In the stomach, NIS protein was immunolocalized in the basolateral membrane of mucin-secreting epithelial cells (see Fig. 14; Refs. 14, 158). However, other investigators (159) have observed NIS-specific immunostaining of the parietal cells. Our group observed immunoreactivity of anti-NIS Ab with an approximately 100-kDa gastric polypeptide, which upon deglycosylation migrated, too, at approximately 50 kDa (14). In all likelihood, these polypeptides correspond, respectively, to glycosylated and nonglycosylated gastric NIS. As with mg-NIS, cyanogen bromide treatment of rat thyroid NIS and gastric NIS proteins yielded the same peptide map (14). This is in agreement with the identity between human thyroid NIS and mg-NIS predicted by the cloning of hNIS cDNAs from gastric mucosa by Spitzweg et al. (154).

    fig.ommittedm6@31, 百拇医药

    Figure 14. Immunohistochemical analysis of NIS protein expression in tissues that exhibit active I^- transport. Middle and upper left panels, Thyroid; upper right panel, salivary gland; bottom left panel, stomach; bottom right panel, lactating mammary gland.m6@31, 百拇医药

    C. Placental NISm6@31, 百拇医药

    The fetal thyroid gland obtains I^- for its own thyroid hormone synthesis from the maternal circulation through the placenta. The expression of the NIS and pendrin genes in the placenta was recently investigated by Bidart et al. (161) by RT-PCR and immunohistochemistry. Expression of both genes was detected by RT-PCR, although to a lesser extent than in the thyroid. Whereas placental NIS gene expression remained unchanged during pregnancy, the pendrin transcript was higher at the end of pregnancy. The pendrin protein was mainly localized by immunohistochemistry in the brush border of the villous syncytiotrophoblast cells, which are in direct contact with the maternal blood (161). In contrast, NIS was immunolocalized only in the cytotrophoblast cells in a nonpolarized fashion, i.e., it was present throughout the plasma membrane. Based on this immunohistochemical localization of NIS and pendrin, it is difficult to explain how I^- is translocated from the maternal blood to the fetal circulation. The same authors analyzed the levels of NIS and pendrin transcripts during in vitro syncytiotrophoblast differentiation 72 h after culturing villous cytotrophoblast cells. In these experiments, NIS transcript level was higher in the cytotrophoblast compared with the syncytiotrophoblast. In contrast, the pendrin transcript level increased significantly when cytotrophoblasts differentiated into syncytiotrophoblasts. A different group (162) localized NIS protein expression mainly to the apical membrane of trophoblast cells and demonstrated NIS mRNA expression in a choriocarcinoma cell line called JAr. It would be of interest to investigate thoroughly the cellular localization of both proteins in the placenta as well as to assess I^- fluxes.

    D. Kidney NIShm:m, 百拇医药

    The level of a patient’s supply of I^- is routinely assessed by measuring urinary I^- excretion. The mechanism of urinary I^- excretion by the kidney is unknown. Glomerular filtration, tubular secretion, and reabsorption have been suggested as possible mechanisms. The question of whether and where NIS is expressed in the kidney remains unsettled given the contradictory findings obtained so far. Vayre et al. (158) and Lacroix et al. (163) found no NIS expression by immunohistochemistry in human kidney (Table 3), whereas Spitzweg et al. (160) detected full-length hNIS mRNA expression by RT-PCR followed by Southern hybridization in human kidney tissue. NIS protein was found by immunohistochemistry all along the nephron (proximal, distal tubuli, and collecting duct, with more prominent staining in the distal tubular system), except for the glomeruli. In the proximal tubular cells, NIS staining was more prominent at the basolateral membrane, whereas in the distal tubular cells NIS localization was mostly intracellular. Functional NIS protein expression by immunoblot and I^- uptake assay was found in a human kidney epithelial cell line derived from Wilms tumor (160). Evidently, more research is needed to assess the precise role of NIS in the kidney.

    VII. Congenital ITD due to NIS Mutations)c@5{:, http://www.100md.com

    Congenital ITD (OMIM 274400) is an infrequent autosomic recessive condition caused by mutations in NIS. The general clinical picture consists of hypothyroidism (which can be normalized in some cases with high I^- supplementation or L-T₄ substitutive therapy), goiter, low thyroid I^- uptake (as determined by scintigraphy), and low saliva/plasma I^- ratio (167, 168). Even though congenital hypothyroidism by all causes is an infrequent disease (incidence 1:3000–1:4000 in neonates; Ref. 169), it has an irreversible deleterious effect on the development of the newborn, finally resulting in cretinism if untreated. Mutations in thyroid-specific molecules, such as TPO (170, 171), Tg (172, 173), and TSHR (174, 175) have been identified among causes of congenital hypothyroidism. Most recently, NIS mutations have also been demonstrated to cause congenital hypothyroidism. In the absence of a functional NIS molecule, I^- has no access to the thyroid epithelial cells, resulting in decreased thyroid hormone biosynthesis and higher circulating levels of TSH, which in turn stimulate the morphological and biochemical changes in the thyroid that lead to the development of goiter.

    Since the first case of congenital hypothyroidism due to an ITD was described by Federman et al. (176), several explanations have been proposed to better define the nature of the defect. However, the molecular basis of this condition began to be examined only after the cloning of the NIS cDNA (3, 33) and the elucidation of the exon-intron organization of the NIS gene (Ref. 34 and Fig. 7). To date, about 58 cases of ITD, belonging to 33 families, have been reported worldwide (9, 50, 168, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204). Twenty-seven cases from 13 families studied at the molecular level have been shown to have a mutation in NIS. Nine mutations have been identified, namely G93R, Q267E, C272X, T354P, 515X (frame shift), Y531X, G543E, G395R, and V59E (Refs. 45, 46, 47, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198 , Fig. 11, and Table 4). Although the clinical picture and genetic alterations of these patients are well described (see Refs. 9 and 162 for detailed reviews of the clinical cases), the molecular mechanisms underlying the effects of most of these mutations have yet to be elucidated, with the exception of T354P, the most extensively analyzed mutation. A detailed structure/function study of T354P revealed that a hydroxyl group at the ß-carbon of the residue at position 354 is essential for thyroid NIS function (49). In addition, the Q267E mutation has been proposed to impair NIS trafficking, as suggested by flow cytometry experiments (203).

    fig.ommitted17h4^, 百拇医药

    Figure 11. Localization of ITD-causing mutations in the NIS protein. Transmembrane segments are represented by cylinders and numbered with Roman numerals. The three glycosylation sites are represented by branches. The nine identified ITD-causing NIS mutations are shown: the letter before the number indicates the original amino acid and the letter after the number indicates the substitution. Amino acids are indicated with the single-letter code. X, Stop codon; fS, frame shift.17h4^, 百拇医药

    fig.ommitted17h4^, 百拇医药

    Table 4. Summary of ITD-causing NIS mutations characterized at the molecular level17h4^, 百拇医药

    The ITD-causing G395R NIS mutation was first identified by Kosugi et al. (204). The reported absence of thyroidal I^- uptake in these patients suggested that, at some level, the G395R NIS mutation impairs NIS function. As a corollary, it also suggested that attributes of residue 395, which is located in the putative transmembrane segment X (Fig. 11), may play a significant role in some aspects of NIS activity. Indeed, Kosugi et al. reported a lack of NIS activity at subsaturating external I^- concentrations (10 µM) in COS cells transfected with the mutant G395R NIS cDNA. In addition, these authors indicated that expression of the G395R NIS protein was indistinguishable from wild-type NIS, as suggested by immunoblot and immunofluorescence analyses (not shown in the report). These findings support the notion that the G395R NIS mutation does not interfere with either the biosynthesis of NIS or its targeting to the plasma membrane.

    More recently, our group extended the observations of Kosugi et al. and carried out a detailed study of the mechanism by which the G395R mutation renders NIS nonfunctional (52). We observed that COS cells transiently transfected with G395R NIS cDNA exhibited no I^- uptake activity not only at a subsaturating external I^- concentration (20 µM), as Kosugi et al. (204) had reported, but also at a supersaturating I^- concentration (320 µM). We also demonstrated by immunoblot analysis that the levels of expression of both the partially and fully glycosylated species of G395R NIS were identical with wild-type NIS, and we showed by both immunofluorescence analysis and surface biotinylation that G395R NIS is properly targeted to the plasma membrane. This is in stark contrast to the reported effects that point mutations have on other transporters, such as the cystic fibrosis transmembrane regulator (205) or SGLT1 (206), in both of which the respective mutations interfere with trafficking of the transporters to the cell surface.

    As the original G395R mutant identified in the patients exhibits no I^- transport activity at any I^- concentration and contains arginine, a positively charged residue with a considerably larger side-chain than glycine, we investigated the effect of size and charge at position 395. We detected no I^- transport activity in any mutant containing a charged residue at position 395 and observed that NIS activity decreased in an inverse relation to the side-chain size of the noncharged residue placed at position 395. Thus, we concluded that the presence of an uncharged amino acid residue with a small side-chain at position 395 is a requirement for NIS function, suggesting that glycine 395 is located in a tightly packed membrane embedded region of NIS. It is clear that the continued study of NIS mutations is likely to lead to the identification of functionally significant residues or segments of NIS..)evn4, 百拇医药

    VIII. NIS in Autoimmune Thyroid Disease (AITD)

    Autoantibodies against the thyroid-specific molecules Tg, TPO, and TSHR are diagnostic markers in AITD. The molecular identification of NIS soon led several groups to investigate the possible role played by NIS in AITD and to attempt to detect the presence of autoantibodies against NIS. Even before the isolation of the NIS cDNA, Raspe et al. (207) found that 1 serum of 147 from patients with AITD inhibited I^- transport activity in primary cultures of dog thyrocytes. Inhibition was specific, given that the serum was still active at 1:1000 dilution and did not inhibit Na⁺/K⁺ ATPase activity. Endo et al. (208) screened sera from patients with AITD by recombinant NIS protein slot-blotted onto nitrocellulose sheets. Sera from 84% of Graves’ disease and 11% of Hashimoto’s thyroiditis patients were positive, but the effect of these positive sera on I^- uptake was not tested. In a subsequent study, the same group (209) concentrated exclusively on Hashimoto’s thyroiditis samples. Eleven percent of the serum samples derived from patients with Hashimoto’s thyroiditis immunoreacted with an approximately 80-kDa polypeptide from FRTL-5 cells. These sera caused 14–62% inhibition of I^- accumulation in CHO cells stably expressing recombinant rNIS. The investigators observed also that some normal sera and patients’ sera that did not immunoreact on immunoblots nevertheless caused approximately 90% inhibition of NIS activity, which was lost after sera were subjected to dialysis.

    Morris et al. (210) synthesized 21 peptides corresponding to putative extracellular segments of rNIS, based on the initial 12-transmembrane-segment secondary structure model proposed for rNIS (3, 4). Serum samples were analyzed by ELISA using the synthetically made peptides. The most highly recognized eight peptides were those corresponding to the fourth, fifth, and sixth extracellular loops and the intracellularly facing COOH terminus of the initial secondary structure model, which correspond, respectively, to the fourth and sixth intracellular and sixth extracellular loops and the intracellularly oriented COOH terminus of the current 13-transmembrane-segment model (Fig. 2). In contrast, none of the control sera displayed any immunoreactivity. The observed recognition of putative intracellular epitopes by these Abs was explained by the investigators as a result of exposure of these internal sequences due to thyroiditis-induced follicular cell damage. No data were provided regarding recognition of the entire NIS molecule by these antisera.

    Ajjan et al. (211) established a CHO cell line stably expressing hNIS devoid of the last 31 amino acids, thus generating a valuable system (CHO-NIS9 cells) for the evaluation of anti-NIS Abs on account of the absence of other thyroid-specific antigens. Eighty-eight sera from patients with Graves’ disease were tested for their effect on I^- uptake. Twenty-seven of 88 (30.7%) of the Graves’ disease sera (and also their corresponding purified IgGs), but none of the controls, inhibited I^- uptake. The auto-Abs were not immunoreactive in immunoblot experiments using extracts from the same cells, an observation that may relate to antigen concentration and/or the absence of linear epitopes in NIS.;k, 百拇医药

    The same authors then established a direct binding assay (212). Serum samples were assessed for their ability to precipitate in vitro-transcribed and -translated S³⁵-labeled hNIS protein. By this method, 22% of Graves and 24% of Hashimoto sera were found to contain NIS-binding antibodies. Seventy-three percent and 43% of the NIS Ab-positive Graves and Hashimoto sera exhibited I^- uptake inhibition in hNIS-transfected CHO cells. Chin et al. (213) screened 514 serum samples from normal subjects and patients with AITD, nonimmune thyroid disease, and nonthyroid autoimmune diseases. Their screening method consisted of assaying for I^- uptake inhibiting activity in a COS cell line stably transfected with hNIS. Although initially these investigators detected some inhibitory activity, after dialysis or IgG purification the I^- uptake inhibitory activity of all samples was lost. Tonacchera et al. (214) also reported some inhibition of I^- accumulation in CHO cells transfected with hNIS by whole sera from patients with Hashimoto’s or Graves’ disease, as well as sera from normal subjects, but the inhibitory effect was similarly lost after sera dialysis. Both of these studies indicate that the inhibition was not mediated by anti-NIS auto-Abs but was, rather, due to unknown factors present in the sera.

    Seissler et al. (215) used a direct immunoprecipitation assay of in vitro-transcribed and -translated [³⁵S]methionine-labeled hNIS molecules. Using a stringent cut-off criterion (99.4th percentile of normal controls), anti-hNIS antibodies were found in only 5.6% of patients with Graves’ disease and 6.9% of patients with Hashimoto’s thyroiditis. These authors therefore reported a lower frequency of anti-hNIS antibodies than that reported previously.?-7|f, 百拇医药

    Kemp et al. (216) used deletion derivatives of the NIS cDNA to identify specific epitopes recognized by anti-hNIS antibodies. Analysis of the results obtained suggested the existence of multiple antibody-binding sites (amino acids 1–134, 191–286, 290–411, and 411–520). The approach taken by the last two groups mentioned, i.e., immunoprecipitation of in vitro-made hNIS, is useful to detect linear epitopes but does not identify conformational epitopes.?-7|f, 百拇医药

    The results obtained thus far are often contradictory. Therefore, the presence of anti-NIS auto-Abs against both linear and conformational epitopes should be pursued. Clearly, a wide range of experimental strategies is necessary to unequivocally determine the existence, real prevalence, functional effects, and possible pathological significance of auto-Abs against NIS in AITD. In summary, although the role of NIS in AITD remains inconclusive, NIS does not seem to play a major role as an autoantigen.

    IX. NIS and Cancerao, 百拇医药

    A. Thyroid cancerao, 百拇医药

    Compared with other cancers, the prevalence of thyroid cancer is relatively low (0.74% in men and 2.3% in women; Ref. 217), and its prognosis is favorable due to the effectiveness of surgical therapy followed by ¹³¹I radioablation and TSH suppression with T₄. Ten-year survival rates for papillary and follicular carcinomas are 95 and 90%, respectively. Unfortunately, the recurrence rate of thyroid cancer is high (~ 30%; Ref. 218), and only one third of patients with distant metastases respond to ¹³¹I therapy with complete remission (219).ao, 百拇医药

    Most thyroid cancers and their metastases exhibit reduced radioiodide accumulation with respect to normal thyroid tissue. Yet, even this reduced I^- transport activity in malignant cells is sufficient for ¹³¹I radioablation to be effective in the majority of cases. In one approach to elucidate the mechanism by which I^- transport activity is decreased in thyroid cancer, Russo et al. (220) analyzed, by direct sequencing after PCR amplification, the NIS cDNA derived from five papillary and two follicular thyroid carcinomas but found no mutations in NIS. In the past, given the reduced radioiodide concentration observed in malignant thyroid tissue, the prevailing expectation was that NIS expression would be decreased in thyroid cancer cells. Since the NIS cDNA and anti-NIS Abs became available, several groups began to test this expectation by investigating NIS expression in human cancerous thyroid epithelial cells. Using RT-PCR, Smanik et al. (33), Ryu et al. (221), Lazar et al. (222), and Park et al. (223) all reported variable or decreased hNIS mRNA expression in papillary carcinomas. Other groups, mindful of the limitations of RT-PCR as a quantitative method, limited their assessment to the presence or absence of NIS transcript in thyroid carcinomas: Arturi et al. (224) found NIS transcript present in 73–96% of differentiated thyroid carcinomas, and Tanaka et al. (225) only in 22% of papillary carcinoma cases. Recently, Arturi et al. (226) reported that 8 of 11 neck lymph node metastases from papillary carcinoma were positive for NIS mRNA, as assessed also by RT-PCR. These results, which vary considerably, should be interpreted knowing that observed changes in NIS mRNA levels do not reflect expression of the NIS protein or its targeting to the plasma membrane. Moreover, the multiple regulatory levels of NIS functional expression (transcriptional, translational, posttranslational, targeting to the plasma membrane, and distribution to intracellular organelles) can lead to widely differing results depending on the technique used and the level at which NIS expression is being assessed. Immunoblot analysis offers the advantages over RT-PCR in that it is a quantitative assay and it detects NIS protein rather than NIS mRNA. Immunohistochemistry also has several advantages over RT-PCR: immunohistochemistry can be performed on archival tissue, requires a small amount of sample tissue, is suitable for the study of consecutive sections of the same sample with different Abs, reflects expression of the NIS protein (not NIS mRNA), and provides crucial information on NIS subcellular localization. In addition, immunohistochemistry allows for the analysis of both the cancerous and surrounding normal tissue from the same specimen, and both tissues can be processed simultaneously.

    Saito et al. (92) carried out both Northern blot and immunoblot analysis of the same papillary carcinomas and compared the results to controls taken from contralateral normal thyroid tissue in four cases. They found increased NIS expression by both methods in three of the cases and similar NIS expression in one papillary carcinoma as compared with the normal thyroid tissue derived from the same thyroid gland. Saito et al. (92) analyzed additional specimens only by immunoblot or immunohistochemistry and found increased NIS protein expression in 7 of 17 papillary carcinomas and abundant NIS staining in 8 of 12 papillary carcinomas by immunohistochemistry. In contrast, NIS protein expression was barely detected in the paratumoral (juxtatumoral, adjacent, or extratumoral) normal tissue. Whereas the findings of Saito et al. show that many thyroid cancers overexpress rather than underexpress NIS, other investigators using immunohistochemistry to detect NIS protein in differentiated thyroid cancers have reported absent (157) or intermediate staining for NIS (227) or just a smaller number of NIS-positive cells in differentiated thyroid cancers than in the surrounding normal tissue (156). All reports in which immunohistochemistry was used describe the NIS immunohistochemical pattern in differentiated thyroid cancer as strongly resembling normal thyroid tissue: NIS expression was heterogenous, as not all follicles or all cells within the same follicle expressed NIS. In addition, NIS was mostly localized on the basolateral membrane of the epithelial cells. Caillou et al. (156) and Castro et al. (227) have also described basolateral localization of NIS in thyroid cancer cells but did not comment on whether these tumor cells retained their polarity. Remarkably, earlier investigations of Na⁺/K⁺-ATPase localization have shown that malignantly transformed thyroid epithelial cells lose their polarity (228). Saito et al. (92) indicated that NIS immunohistochemistry staining was present throughout the cell except in the nuclear area. In two other reports (156, 222), the expression of the TSHR and NIS was investigated simultaneously in thyroid cancer by RT-PCR and immunohistochemistry. The TSHR was normally expressed (quantitatively) in most of the tumors, whereas NIS expression was found to be decreased in all tumors by both methods (156, 222). The localization of the TSHR was not described, even though the TSHR has previously been reported to be localized in thyroid cancer cells both in the basolateral surface of the plasma membrane and intracellularly. In normal cells, the TSHR is localized exclusively in the basolateral side of the plasma membrane. Loss of polarization and impaired membrane targeting of other membrane proteins have also been observed in malignant thyroid epithelial cells (229). In thyroid carcinomas the epidermal growth factor receptor, as detected by immunohistochemistry, was overexpressed and localized not only pericellularly but also and mostly intracellularly, rather than exclusively in the basolateral membrane as in normal cells, whereas the levels of epidermal growth factor receptor mRNA were found to be similar in normal and cancerous tissues. Therefore, a thorough evaluation of the expression of a given molecule in cancerous cells must include determinations of the molecule’s transcript, protein, and cellular distribution.

    More recently, seeking to clarify the reported variability of immunohistochemistry results, we analyzed NIS protein expression in 57 thyroid cancer samples (i.e., a much larger number of samples than any of the preceding studies) by immunohistochemistry using high-affinity anti-NIS Abs (93). We found that, far from lacking expression, as many as 70% of the studied thyroid cancer samples overexpressed NIS compared with the surrounding normal tissue. The immunohistochemical localization of NIS was mostly intracellular; in a few cases, distinct plasma membrane staining was observed. When plasma membrane staining was present, it was not polarized, i.e., it was visible in both the basolateral and apical surfaces of the cell. Therefore, we found that the decrease in I^- uptake in most thyroid carcinomas is not due to low NIS expression but to alterations in NIS trafficking.k}4, 百拇医药

    NIS must be expressed, targeted, and retained in the appropriate plasma membrane surface in polarized epithelial thyroid cells for active I^- transport to occur. As indicated in Section IV.A, TSH regulates NIS distribution between the plasma membrane and intracellular membrane compartments. In thyroid cancer cells, I^- transport can still be present even in the absence of cell polarization, but targeting to and retention in the plasma membrane remain essential if active I^- transport is to take place. Furthermore, Tonacchera et al. (230) reported recently that 54% of benign nonfunctional thyroid nodules overexpressed hNIS protein, as compared with normal surrounding tissue; significantly, NIS was located intracellularly in these nodules. These results underscore the importance of elucidating the molecular mechanism involved in proper targeting to and retention of NIS at the plasma membrane.

    Some investigators have attempted to induce NIS expression in thyroid carcinoma cell lines with demethylation treatment (231) and retinoic acid (RA) (232). Venkataraman et al. (231) found that the NIS promoter region is strongly methylated in the investigated thyroid carcinoma cell lines. They were able to induce NIS mRNA expression in four human thyroid carcinoma cell lines and restored some I^- uptake activity in two other cell lines using 5-azacytidine and sodium butyrate. Kogai et al. (233) treated four human papillary cell lines lacking NIS expression with a histone deacetylase inhibitor (trichostatin A) and a demethylating agent (5-azacytidine) and found no effect on NIS expression. I^- uptake was restored upon transfection of these cell lines with hNIS cDNA, suggesting that the posttranscriptional machinery governing NIS expression and plasma membrane targeting in these cells was intact. This was not due to mutations in the NIS promoter, given that nuclear extracts from the papillary carcinoma cell lines exhibited reduced binding to the NIS promoter region as compared with FRTL-5 cells. The authors concluded that the absence of NIS expression in the carcinoma cell lines may be due to the lack or diminished expression of a yet unknown transcription factor(s).

    RA treatment was also effective in reinducing NIS expression and I^- uptake in certain thyroid carcinoma cell lines, but its effect and clinical usefulness are still under debate. RAs are biologically active metabolites of vitamin A. Retinol is stored in the liver and circulates in the bloodstream. Upon entering into the cells, retinol is converted into retinal and RA by retinol dehydrogenase and retinal dehydrogenase, respectively. RAs [all-trans RA (tRA), 9-cis RA] bind to nuclear receptors, which behave as ligand-binding transcription factors. RAs have been shown in several cell types to play regulatory roles in cell differentiation. Schmutzler et al. (232) investigated the effect of RA on NIS mRNA and protein levels and on NIS function in various human thyroid carcinoma cell lines and in FRTL-5 cells. In the two human follicular carcinoma cell lines investigated, no NIS transcript was found but, after 24 h of 1 µM tRA treatment, a significant amount of NIS mRNA was detected. Interestingly, both cell lines expressed the same amount of NIS protein when compared with each other with and without tRA treatment, and both cell lines under both conditions exhibited no I^- uptake activity, indicating that the determination of NIS transcript does not reflect the amount, functional activity, and subcellular localization of the NIS protein. In contrast, in FRTL-5 cells tRA treatment caused a significant decrease in NIS transcript, protein level, and I^- uptake activity.

    In conclusion, whereas impaired I^- uptake in differentiated thyroid cancer could result from absent or decreased expression of the NIS gene, in a majority of cases lowered NIS function seems to be due to impaired targeting and/or insufficient retention of NIS in the plasma membrane, even though NIS is mostly overexpressed in these cells. Therefore, improvements in ¹³¹I radioablation therapy might result from both inducing NIS transcription in thyroid cancer cells when NIS is not expressed and promoting NIS targeting to the plasma membrane when it is mostly expressed intracellularly.|h\qn2/, http://www.100md.com

    B. Breast cancer|h\qn2/, http://www.100md.com

    The ability of cancerous thyroid cells to actively transport I^- via NIS provides a unique and effective delivery system to detect and target these cells for destruction with therapeutic doses of radioiodide, largely without harming other tissues. Therefore, it seems feasible that radioiodide could be a diagnostic and therapeutic tool for the detection and destruction of other cancers in which NIS is functionally expressed. Pointing in this direction is our recent report (14) in which we showed that both human breast carcinomas and experimental mammary carcinomas in transgenic mice express NIS. In vivo scintigraphic imaging of experimental mammary adenocarcinomas in nongestational and nonlactating female transgenic mice carrying either an activated ras oncogene or overexpressing the neu oncogene demonstrated pronounced, active, specific, and perchlorate-inhibitable NIS activity (14). Hence, we concluded that transgenic mice bearing experimental mammary tumors provide an excellent model to study the potential role of NIS in mammary cancer and particularly the possible effectiveness of radioiodide therapy in combating this disease. Furthermore, we (14) showed, by immunohistochemistry, that 87% of 23 human invasive breast cancers and 83% of 6 ductal carcinomas in situ expressed NIS, as compared with only 23% of 13 extratumoral samples from the vicinity of the tumors. Even more significantly, none of the eight normal samples from reductive mammoplasties we studied expressed NIS. Kogai et al. (234) reported an increase in NIS mRNA, NIS protein, and I^- uptake activity in a human mammary adenocarcinoma cell line (MCF-7) in response to tRA treatment. We have recently developed a method for early detection (by flow cytometry) of mg-NIS expression in human mammary adenocarcinoma cells collected by fine needle aspiration (Fig. 12). The results obtained with this method correlate closely with mg-NIS expression detected by immunohistochemistry of the corresponding biopsy specimens (Fig. 12).

    fig.ommitted6j][((, http://www.100md.com

    Figure 12. Flow cytometry of human mammary gland tissue. A, Schematic representation of fine-needle-aspirated breast cancer cells expressing mg-NIS (in red) detected by an anti-NIS Ab followed by a fluorescent secondary Ab, the binding of which produces a shift in fluorescence intensity (compare middle and upper panels) that is competed out by excess peptide (lower panel). B, mg-NIS detection by immunohistochemistry in a human mammary adenocarcinoma (upper panel) competed out by excess peptide (lower panel).6j][((, http://www.100md.com

    The above-described results suggest that radioiodide may represent a novel potential alternative diagnostic and therapeutic modality in breast cancer. Moreover, the potential diagnostic value of the high prevalence of mg-NIS expression (80%) in human breast cancer (mg-NIS was virtually absent in normal tissue) becomes apparent when compared with the prevalence (33%) of the main current breast cancer marker Her2/neu. Whereas clinical trials and additional studies are necessary to determine the extent of NIS activity in human breast carcinomas and the efficacy of radioiodide treatment for breast cancer, the possible role of NIS in this disease is one of the most dramatic instances of the medical impact of NIS research. A frequently mentioned possible obstacle to the therapeutic use of radioiodide in extrathyroidal cancers is the widely held notion that radioiodide therapy is unlikely to be effective in nonthyroidal cells that, while functionally expressing NIS (whether endogenously or by targeted transfection), lack the ability to organify I^-. The reasoning is that the absence of organification might result in the isotope not being retained in the cells for a sufficiently long time. Yet, in studies by Spitzweg and colleagues (235, 236, 237) on NIS-expressing prostate cancer cells (see next section), radioiodide treatment was effective even in the absence of I^- organification. Moreover, no data are available to indicate that I^- organification is required for radioiodide therapy to be effective. Thyroid cancer metastases often display a disrupted follicular architecture and lack Tg expression, both indicative of absence of I^- organification, and yet radioiodide therapy against these metastases is effective. Therefore, the lack of I^- organification is not necessarily the obstacle that many researchers expected.

    X. NIS in Gene Transferdza$], http://www.100md.com

    As indicated in Section IX, for several decades NIS has played a key role in the diagnosis and treatment of differentiated thyroid carcinomas and their metastases. The functional expression of NIS in these tumors and their metastases makes them susceptible to therapeutic destruction with radioiodide, thus improving significantly the prognosis of thyroid cancer patients. The success of radioiodide therapy is compelling: the mortality of patients with thyroid cancer who are treated with ¹³¹I is just 3%, as opposed to 12% for those who are not treated (238). The effect of ¹³¹I is proportional to the effective radiation dose delivered to the tumor tissue, which depends on the effective half-life of ¹³¹I in the tumor and the cells’ ¹³¹I concentrating ability. The latter in turn depends on the rates of ¹³¹I influx and efflux. It is clearly of major significance that radioiodide therapy is remarkably free of serious adverse effects, except for transient and usually mild sialadenitis and reversible myelosuppression (239). The cloning and characterization of NIS, in light of the ample experience accumulated over the last 60 yr of treating thyroid patients with radioiodide, has led to the development of a novel gene therapy strategy against cancer: the targeted expression of functional NIS molecules to cancer cells aimed at rendering them susceptible to destruction with radioiodide. Several in vitro experiments on NIS-based gene therapy for both diagnostic and therapeutic purposes have been reported in which NIS-mediated radioiodide uptake was used to visualize and destroy malignant tumor cells. Shimura et al. (240) transfected the hNIS gene into malignant rat thyroid cells that did not concentrate I^-, resulting in increased I^- accumulation in these cells in vitro. Using a retroviral vector, Mandell et al. (241) have recently introduced rNIS into melanoma, ovarian, liver, and colon carcinoma cells. The resulting rNIS-transduced tumor cells exhibited I^- uptake activity. In vitro experiments showed that these transduced cells could be destroyed by accumulation of ¹³¹I. Cho et al. (242) have recently shown, using NIS-containing recombinant adenovirus, that hNIS can be functionally expressed in xenografted human glioma.

    NIS gene therapy using tissue-specific promoters provides a way to selectively target NIS to malignant cells, maximizing tissue-specific cytotoxicity and minimizing toxic side-effects in nonmalignant cells. Spitzweg et al. (235) induced tissue-specific androgen-dependent I^- uptake activity in prostate cancer cells by prostate-specific antigen promoter-directed NIS expression in vitro. Subsequently, these authors established xenografts in nude mice from a NIS-expressing human prostate cancer cell line that actively accumulated in vivo as much as 25–30% of administered I^- (236). Strikingly, the size of the xenograft tumors in these mice was significantly reduced after a single ip injection of a therapeutic dose (3 mCi) of ¹³¹I (236). Confirming and extending these results, Spitzweg et al. (237) then applied a novel form of gene therapy using adenovirus-mediated in vivo NIS gene transfer followed by ¹³¹I administration for treatment of prostate cancer. They demonstrated pronounced radioiodide uptake in prostate cancer xenografts in nude mice injected with an adenovirus carrying the NIS gene linked to the cytomegalovirus promoter. Moreover, these authors observed an average tumor volume reduction of 84 ± 12% upon administration of 3 mCi of ¹³¹I, demonstrating that in vivo NIS gene delivery into nonthyroidal tumors can lead to sufficient NIS activity for therapeutic radioiodide doses to be effective. Because there is no I^- organification in NIS-expressing prostate cancer cells, as pointed out in the preceding section, these results provide strong evidence against the concept that I^- organification is a requirement for the effectiveness of radioiodide therapy.

    Although specific, safe, and efficient gene-delivery systems still have to be examined further, the gene therapy approach is undoubtedly one of the most promising developments concerning the possible uses of the molecular characterization of NIS in the diagnosis and treatment of cancer in a wide variety of tissues.3, 百拇医药

    XI. Concluding Remarks3, 百拇医药

    NIS research has clearly become an exciting field in its own right. The many studies on NIS spurred by isolation of the rNIS cDNA have been far-reaching, extending from detailed structure/function analysis of the molecule and elucidation of NIS regulatory processes at several levels to novel medical applications. Investigations on NIS topology and on the functional role of specific amino acid residues have yielded significant structure/function information. Kinetic and electrophysiological studies have led to a mechanistic transport model for NIS, proposing that Na⁺ binds before I^- (with a 2:1 Na⁺/I^- stoichiometry), then the NIS-Na₂I complex is formed, and a conformational change in NIS exposes the bound Na⁺ and I^- ions to the interior of the cell and releases them (Fig. 13), so that after an ordered and sequential binding, transport of both ions is simultaneous. Current and future mechanistic experiments on NIS will likely provide insight into a fundamental process in biology, i.e., the mechanism by which the energy stored in an ion chemical gradient (the Na⁺ concentration gradient) is transduced into work (i.e., active transport of I^-). Findings obtained from such studies will also be applicable to many prokaryotic and eukaryotic transporters.

    fig.ommitted8-l$g, 百拇医药

    Figure 13. Schematic representation of a NIS mechanistic model. The kinetic data suggest that both Na⁺ ions bind to NIS before I^- (A"->"8-l$g, 百拇医药

    B"->"8-l$g, 百拇医药

    C). In the presence of I^-, the complex NIS-Na₂I is formed (symport mode) (C"->"8-l$g, 百拇医药

    D), which undergoes a conformational change to expose the bound two Na⁺ and I^- ions to the interior of the cell (D"->"8-l$g, 百拇医药

    E). Both Na⁺ ions and I^- are released into the cytoplasmic compartment (E"->"8-l$g, 百拇医药

    F"->"8-l$g, 百拇医药

    G"->"8-l$g, 百拇医药

    H), and the empty carrier (H) undergoes another conformational change to expose the binding sites to the external solution again (A). Charge movement data suggest that the Na⁺ binding dissociation does not contribute greatly to the total observed charge. Thus, it is proposed that NIS charge movements arise primarily from conformational changes of the empty carrier (H"->"

    A). In the Na⁺ uniport mode (B"->"*kefdj1, 百拇医药

    G), one Na⁺ ion binds to NIS (A"->"*kefdj1, 百拇医药

    B) and may cross the membrane via NIS. Release of Na⁺ into the cytoplasm (G"->"*kefdj1, 百拇医药

    H) is followed by the return of the empty binding site to complete the pathway (H"->"*kefdj1, 百拇医药

    A). For more details refer to Ref. 32 .*kefdj1, 百拇医药

    Even before its molecular characterization, NIS was extensively and successfully used in the management of thyroid disease. Now our conception of NIS has fast evolved from viewing it as an elusive thyroid-specific marker, almost a missing link in the thyroid puzzle, to understanding it as a versatile multifaceted molecule that is expressed and regulated differently in several extrathyroidal tissues (Fig. 14) and in breast cancer. The sole prospect of relying on NIS to diagnose and treat breast cancer with radioiodide has already brought the medical expectations of NIS research to a new level. It seems safe to predict that the continued study of the mechanisms involved in NIS biogenesis, regulation, subcellular distribution, and function will considerably extend both the basic and clinical impact of NIS.

    "" hspace=57\8eh, http://www.100md.com

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