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Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
Correspondence: Address all correspondence and requests for reprints to: Nancy Carrasco, M.D., Department of Molecular Pharmacology, Albert Einstein College of Medicine, Forchheimer Building, Room 209, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: carrasco{at}aecom.yu.edu
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Abstract |
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 Top Abstract I. Introduction and BackgroundII. Molecular Characterization...III. Transcriptional Regulation...IV. Regulation of NIS...V. Signal TransductionVI. Extrathyroidal NIS...VII. Congenital ITD due...VIII. NIS in Autoimmune...IX. NIS and CancerX. NIS in Gene...XI. Concluding RemarksReferences |
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I. Introduction and Background |
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TopAbstractI. Introduction and BackgroundII. Molecular Characterization...III. Transcriptional Regulation...IV. Regulation of NIS...V. Signal TransductionVI. Extrathyroidal NIS...VII. Congenital ITD due...VIII. NIS in Autoimmune...IX. NIS and CancerX. NIS in Gene...XI. Concluding RemarksReferences |
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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.
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).
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). 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.
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II. Molecular Characterization of NIS |
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TopAbstractI. Introduction and BackgroundII. Molecular Characterization...III. Transcriptional Regulation...IV. Regulation of NIS...V. Signal TransductionVI. Extrathyroidal NIS...VII. Congenital ITD due...VIII. NIS in Autoimmune...IX. NIS and CancerX. NIS in Gene...XI. Concluding RemarksReferences |
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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).
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and 4).
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]. 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).
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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.
C. The road to NIS characterization
1. N-linked glycosylation of NIS: implications for the NIS secondary structure model.
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.
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.
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.
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).
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4. Electrophysiological analysis of NIS: mechanism, stoichiometry, and specificity.
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.
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). 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 (Ki = 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.
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.
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III. Transcriptional Regulation of NIS |
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TopAbstractI. Introduction and BackgroundII. Molecular Characterization...III. Transcriptional Regulation...IV. Regulation of NIS...V. Signal TransductionVI. Extrathyroidal NIS...VII. Congenital ITD due...VIII. NIS in Autoimmune...IX. NIS and CancerX. NIS in Gene...XI. Concluding RemarksReferences |
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is a schematic representation of the rNIS promoter structure.
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IV. Regulation of NIS Expression and Function |
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TopAbstractI. Introduction and BackgroundII. Molecular Characterization...III. Transcriptional Regulation...IV. Regulation of NIS...V. Signal TransductionVI. Extrathyroidal NIS...VII. Congenital ITD due...VIII. NIS in Autoimmune...IX. NIS and CancerX. NIS in Gene...XI. Concluding RemarksReferences |
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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.
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-aminobutyric transporter, prevents its internalization from the basolateral surface of polarized epithelial cells (87). 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.
B. Posttranscriptional regulation of NIS
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–
1. Recent research on the Wolff-Chaikoff effect.
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 
-lactone and -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).
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.
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.
2. Stunning.
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 NIS
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-, TNF-ß, interferon- (IFN-), IL-1, IL-1ß, IL-6, and TGF-ß1, all of which exerted an inhibitory effect on thyroid function, including decreased NIS expression and I– uptake.
Ajjan et al. (115) and Spitzweg et al. (111) have reported that, in FRTL-5 cells, TNF 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 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- 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- on thyroid function in vivo in transgenic mice expressing IFN- in the thyroid. IFN- 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 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, 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
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. Estradiol
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.
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V. Signal Transduction |
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TopAbstractI. Introduction and BackgroundII. Molecular Characterization...III. Transcriptional Regulation...IV. Regulation of NIS...V. Signal TransductionVI. Extrathyroidal NIS...VII. Congenital ITD due...VIII. NIS in Autoimmune...IX. NIS and CancerX. NIS in Gene...XI. Concluding RemarksReferences |
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