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Endocrine Reviews 24(1):48 –77
Copyright © 2003 by The Endocrine Society
doi: 10.1210/er.2001-0029
The Sodium/Iodide Symporter (NIS): Characterization,
Regulation, and Medical Significance
ORSOLYA DOHÁN, ANTONIO DE LA VIEJA, VIKTORIYA PARODER, CLAUDIA RIEDEL,
MONA ARTANI, MIA REED, CHRISTOPHER S. GINTER, AND NANCY CARRASCO
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
The Naⴙ/Iⴚ symporter (NIS) is an integral plasma membrane
glycoprotein that mediates active Iⴚ transport into the thyroid follicular cells, the first step in thyroid hormone biosynthesis. NIS-mediated thyroidal Iⴚ 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 Iⴚ transport in other tissues, including salivary glands, gastric mucosa, and lactating mammary
gland, in which it translocates Iⴚ 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 eluci-
dation 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 Iⴚ 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. (Endocrine Reviews
24: 48 –77, 2003)
I. Introduction and Background
II. Molecular Characterization of NIS
A. Summary of the molecular characteristics of NIS
B. NIS protein family
C. The road to NIS characterization
III. Transcriptional Regulation of NIS
IV. Regulation of NIS Expression and Function
A. TSH
B. Posttranscriptional regulation of NIS
C. Regulation of NIS activity by I⫺
D. Effect of cytokines on NIS
E. Tg
F. Estradiol
V. Signal Transduction
VI. Extrathyroidal NIS Expression
A. Mammary gland NIS (mg-NIS)
B. NIS in the gastrointestinal tract
C. Placental NIS
D. Kidney NIS
VII. Congenital ITD due to NIS Mutations
VIII. NIS in Autoimmune Thyroid Disease (AITD)
IX. NIS and Cancer
A. Thyroid cancer
B. Breast cancer
X. NIS in Gene Transfer
XI. Concluding Remarks
I. Introduction and Background
T
HE 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 iodinecontaining 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
Abbreviations: Ab, Antibody; AITD, autoimmune thyroid disease;
CHO, Chinese hamster ovary; hNIS, human NIS; IFN-␥, interferon-␥;
ITD, iodide transport defect; mg-NIS, mammary gland NIS; MMI,
1-methyl-2-mercaptoimidazole; MV, membrane vesicles; NIS, sodium/
iodide symporter; NPT, sodium/proline cotransporter; PKA, protein
kinase A; PTU, 6-n-propyl-2-thiouracil; RA, retinoic acid; rNIS, rat NIS;
Tg, thyroglobulin; TPO, thyroid peroxidase; SGLT, sodium/glucose cotransporter; SMIT, sodium/myo-inositol cotransporter; SMVT, sodium/multivitamin transporter; tRA, all-trans RA; TSHR, TSH receptor.
48
⫹
Dohán et al. • The Sodium/Iodide Symporter
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 –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.
17–19 for reviews). Briefly (Fig. 1), NIS-mediated I⫺ accumulation in the thyroid is an active transport process that
Endocrine Reviews, February 2003, 24(1):48 –77
49
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-2mercaptoimidazole (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 NISmediated I⫺ uptake, are stimulated by TSH. In contrast, I⫺
accumulation in extrathyroidal tissues is not regulated by
TSH (1).
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.
II. Molecular Characterization of NIS
A. Summary of the molecular characteristics of NIS
Membrane proteins in general and membrane transport
proteins in particular have not been amenable to highresolution 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.
23–27). 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 en-
50
Endocrine Reviews, February 2003, 24(1):48 –77
Dohán et al. • The Sodium/Iodide Symporter
FIG. 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.
TABLE 1. Epidemiological impact of iodine deficiency disorders
(IDD)
● 1.6 billion people worldwide are at risk of IDD (⬃30% of the
world’s population)
● 655 million people suffer from goiter
● 50 million children suffer from varying degrees of IDD
● 43 million have IDD-related brain damage and mental
retardation
● 11 million are overt cretins, the most severe form of IDD
Data from World Health Organization (http://www.who.int/nut/
idd.htm), International Council for the Control of Iodine Deficiency
Disorders (http://www.people.virginia.edu/⬃jtd.iccidd/), and United
Nations Children’s Fund (http://www.unicef.org).
coding 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 (Kd ⬃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
Dohán et al. • The Sodium/Iodide Symporter
Endocrine Reviews, February 2003, 24(1):48 –77
51
FIG. 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.
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 freezefracture 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).
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).
B. NIS protein family
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.
52
Endocrine Reviews, February 2003, 24(1):48 –77
Dohán et al. • The Sodium/Iodide Symporter
FIG. 3. NIS cDNA and protein sequence identity in four species: human, pig, rat, and mouse. ORF, Open reading frame.
However, some important differences in cation selectivity
and stoichiometry exist (see Section II.C.4).
Studies carried out in several members of the family
(30, 38 – 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.
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 Nlinked 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-transmembranesegment 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 FLAGcontaining 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 NNSScontaining NIS into COS cells, thus proving that the NH2
Dohán et al. • The Sodium/Iodide Symporter
Endocrine Reviews, February 2003, 24(1):48 –77
53
FIG. 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.
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
54
Endocrine Reviews, February 2003, 24(1):48 –77
Dohán et al. • The Sodium/Iodide Symporter
FIG. 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.
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 ␤-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 hydroxylcontaining 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).
Dohán et al. • The Sodium/Iodide Symporter
Endocrine Reviews, February 2003, 24(1):48 –77
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.
FIG. 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.
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.
5. Effects of perchlorate (ClO4⫺) and thiocyanate (SCN⫺) on NIS
function. 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
TABLE 2. Kinetic parameters of NIS in various systems
Thyroid slices
Km I⫺ (␮M)
30
FRTL-5 cells
FRTL-5 MV
31
20
Symporter
Km
(mM)
Na⫹
Hill coeff.
␶Symporter (sec
Thyroid MV
4.8, 5
K
1.6, 1.8
2
)
Km SCN (␮M)
Ki ClO4⫺ (␮M)
Refs.
35
ⱖ2
74
22
Hill coeff.
⫺
NIS in CHO cells
(patch-clamp)
36
(mM)
Ki SCN (␮M)
36, 33
28
␶Uniporter (sec⫺1)
⫺
NIS in oocytes
70
⫺1
Uniporter
m Na⫹
1
30
15.3
20 –30
15.9
96
2
19
63
55
82
11.0
1.8
1.5
243, 244
3, 32
61, 62
56
Endocrine Reviews, February 2003, 24(1):48 –77
the Ki 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 (Ki ⫽ 1.7 ␮m) 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 amiodaroneinduced 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-␮g/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 (55–57). 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 PTUblocked 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 radio-
Dohán et al. • The Sodium/Iodide Symporter
FIG. 7. Substrate selectivity of NIS. Inward currents induced by various
anions (500 ␮M) were recorded at Vm ⫽ ⫺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⫺, SO32⫺, CO32⫺, S2O32⫺, and HPO42⫺.
Data are reported as mean ⫾ SE (n ⫽ 3). Adapted from Ref. 32.
activity in the cytosol of thyrocytes, given that chlorate is
readily translocated via NIS into the cell. 36Cl-Chlorate is a
36
Cl 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.
III. Transcriptional Regulation of NIS
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 (65–74). These findings have
been extensively reviewed elsewhere (7–13, 65–74). Figure 8 is
a schematic representation of the rNIS promoter structure.
IV. Regulation of NIS Expression and Function
A. TSH
TSH is an approximately 30-kDa glycoprotein biosynthesized in the adenohypophysis by basophilic cells known as
Dohán et al. • The Sodium/Iodide Symporter
Endocrine Reviews, February 2003, 24(1):48 –77
57
FIG. 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.
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␣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 dis-
appeared 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 follicleforming 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
58
Endocrine Reviews, February 2003, 24(1):48 –77
(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.
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 – 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 proteinprotein interactions. PDZ target sequences are recognized by
PDZ binding proteins (84). Among the various proteins that
Dohán et al. • The Sodium/Iodide Symporter
contain PDZ domains (84) is PDZ binding protein LIN-7,
which, by recognizing a PDZ target motif in the epithelial
␥-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).
FIG. 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).
Dohán et al. • The Sodium/Iodide Symporter
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 –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 WolffChaikoff 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
Endocrine Reviews, February 2003, 24(1):48 –77
59
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 dosedependent 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-8,11,14eicosa-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 TSHstimulated 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 downregulation 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
60
Endocrine Reviews, February 2003, 24(1):48 –77
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 dosedependent 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 halflife 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
Dohán et al. • The Sodium/Iodide Symporter
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-␤, interferon-␥ (IFN-␥), IL-1␣, IL-1␤, IL-6, and TGF-␤1, 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 -␤ were also studied in human thyroid cells in culture, in which a dosedependent decrease of cAMP levels and Tg expression was
observed (118). This effect was enhanced when TNFs were
added together with IL-1␤ (111).
TGF-␤ had a similar effect to TNF, i.e., it also inhibited I⫺
uptake and NIS mRNA in a time- and dose-dependent manner. TGF-␤ 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-␤ 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-␤ 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.
Dohán et al. • The Sodium/Iodide Symporter
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-1␤, 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-1␤
(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-␤,
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-1␤ 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 TSHincreased 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).
Endocrine Reviews, February 2003, 24(1):48 –77
61
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.
V. Signal Transduction
Hormones and growth factors exert their effects on thyroid
cells via several signal transduction pathways. The TSHTSHR-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 –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 (131–133). Interestingly, recent evidence indicates that
cAMP pathways, both PKA dependent and independent,
contribute to thyroidal cell differentiation and therefore NIS
expression (134 –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 Expression
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⫺
62
Endocrine Reviews, February 2003, 24(1):48 –77
Dohán et al. • The Sodium/Iodide Symporter
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, 153–155, 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.
(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
TABLE 3. NIS expression and activity in different tissues
Activity
Thyroid gland
Salivary glands
Gastric mucosa
Lactating mammary gland
Adrenal gland
Appendix
Bladder
Brain
Choroid plexus
Ciliary body of the eye
Colon
Esophagus
Extraocular fat
Extraocular muscle tissue
Heart
Kidney
Liver
Lung
Mammary gland
Muscle
Orbital fibroblast
Ovary
Pancreas
Pituitary
Placenta
Prostate
Rectum
Skin
Small intestine
Spleen
Testis
Thymus
Tong
Uterus
⫹,
⫹,
⫹,
⫹,
a
a
a
a, j, q
⫹, a
⫹, a
NB
⫹,
⫹,
⫹,
⫹,
b,
b,
b,
b,
c, o
c
o
o
RT-PCR
⫹,
⫹,
⫹,
⫹,
⫹,
WB
c, d, n, o
b, c, n
b, c, d, j, n, o
o, j
c
⫺, o
⫹, j
⫹, j
⫹, j, q
⫹, o
⫹, d
⫹, b
,c
⫹, i
⫹, c, n
⫹, b, c, d
⫹,
⫹,
⫹,
⫹,
⫹,
c, o
c
c
k, l
c, o
⫹,
⫹,
⫹,
⫹,
⫹,
o
o
o
c, o
c
⫹, c
⫺, b, c
⫺, b
⫺, b
⫺, o
⫺, o
⫺, o
⫺, c
⫺, b
⫺, j
⫺, j
⫺, j
IH
⫹,
⫹,
⫹,
⫹,
e, f, g, h, j, m, n
f, g, j, n
g, h, j, n
j, q
⫺, n
⫺, g
⫺, h
⫹, h, n
⫺, g, n
⫺, g
⫹, i
⫺, g, n
⫺, h
⫹, g
⫺, j, n
⫺, h
⫹, h
⫺, g, n
⫹, k, l
⫺, c
⫺, o
⫹, g
⫹, p
⫺, o
NB, Northern blot; WB, Western blot; IH, immunohistochemistry; ⫹, positive; ⫺, negative.
Refs.: a, 152; b, 153; c, 154; d, 34; e, 156; f, 157; g, 158; h, 159; i, 160; j, 14; k, 161; l, 162; m, 93; n, 163; o, 36; p, 164; q, 155.
⫺, g
⫺, n
⫺, n
⫺, g
⫺, h
⫺, h, n
Dohán et al. • The Sodium/Iodide Symporter
Endocrine Reviews, February 2003, 24(1):48 –77
63
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
␤-estradiol, oxytocin, and PRL led to the highest level of NIS
expression.
FIG. 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 ␮g/ml of affinity-purified anti-hNIS
Ab, followed by 0.3 ␮g/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 ␮g); 2, breast adenocarcinoma (100 ␮g); 3, mixed tumor of the salivary gland (20 ␮g); 4,
gastric mucosa (20 ␮g); 5, multinodular goiter (20 ␮g); 6, thyroid
follicular adenoma (20 ␮g); 7, thyroid papillary carcinoma (follicular
variant, 20 ␮g); 8, thyroid from a patient with Graves’ disease (8 ␮g);
9, hNIS stably transfected Madin-Darby kidney epithelial cells
(MDCK) (5 ␮g).
A. Mammary gland NIS (mg-NIS)
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 highaffinity 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 antiNIS 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⫺
B. NIS in the gastrointestinal tract
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).
C. Placental NIS
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
64
Endocrine Reviews, February 2003, 24(1):48 –77
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 NIS
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
Congenital ITD (OMIM 274400) is an infrequent autosomic
recessive condition caused by mutations in NIS. The general
Dohán et al. • The Sodium/Iodide Symporter
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 –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– 47, 189 –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 mech-
FIG. 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.
Dohán et al. • The Sodium/Iodide Symporter
Endocrine Reviews, February 2003, 24(1):48 –77
65
TABLE 4. Summary of ITD-causing NIS mutations characterized at the molecular level
Mutation
V59E
G93R
Q267E
C272X
T354P
G395R
fS515X
G543E
a
Exon
Localization
1
1
6
6
8
10
Intron 12
13
TMS II
TMS III
iS-8
iS-8
TMS IX
TMS X
eS-13
TMS XIII
Trafficking defect
Structural requirements
Yes
No
No
Yesa
Hydroxyl group at the ␤-carbon
Small neutral side-chain
Ref.
202
198
200, 203
199, 201, 203
49, 195, 196, 197, 198
52, 204
200, 203
198
De la Vieja et al. (unpublished observation).
anisms 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
␤-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).
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 ␮m) 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 ␮m),
as Kosugi et al. (204) had reported, but also at a supersaturating I⫺ concentration (320 ␮m). 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.
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 second-
66
Endocrine Reviews, February 2003, 24(1):48 –77
ary 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 thyroiditisinduced 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.
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]methioninelabeled 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.
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
Dohán et al. • The Sodium/Iodide Symporter
of in vitro-made hNIS, is useful to detect linear epitopes but
does not identify conformational epitopes.
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 Cancer
A. Thyroid cancer
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).
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
Dohán et al. • The Sodium/Iodide Symporter
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 NISpositive 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
Endocrine Reviews, February 2003, 24(1):48 –77
67
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.
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
68
Endocrine Reviews, February 2003, 24(1):48 –77
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 ␮m 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.
B. Breast cancer
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
Dohán et al. • The Sodium/Iodide Symporter
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).
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 (235–237) on NISexpressing 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 Transfer
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 ra-
Dohán et al. • The Sodium/Iodide Symporter
Endocrine Reviews, February 2003, 24(1):48 –77
69
FIG. 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).
dioiodide, 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 tissuespecific 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
70
Endocrine Reviews, February 2003, 24(1):48 –77
Dohán et al. • The Sodium/Iodide Symporter
FIG. 13. Schematic representation of a NIS mechanistic model. The kinetic data suggest that both Na⫹ ions bind to NIS before I⫺ (A3 B3 C).
In the presence of I⫺, the complex NIS-Na2I is formed (symport mode) (C3 D), which undergoes a conformational change to expose the bound
two Na⫹ and I⫺ ions to the interior of the cell (D3 E). Both Na⫹ ions and I⫺ are released into the cytoplasmic compartment (E3 F3 G3 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 (H3 A). In the Na⫹ uniport mode (B3 G), one Na⫹
ion binds to NIS (A3 B) and may cross the membrane via NIS. Release of Na⫹ into the cytoplasm (G3 H) is followed by the return of the empty
binding site to complete the pathway (H3 A). For more details refer to Ref. 32.
FIG. 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.
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 adminis-
tration 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 pros-
Dohán et al. • The Sodium/Iodide Symporter
tate 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.
XI. Concluding Remarks
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.
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.
Acknowledgments
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: [email protected]
This work was supported in part by the Army Breast Cancer Research
Program (to O.D.); the Spanish Ministry of Education and Culture (to
A.D.V); and NIH Grant DK-41544, the Susan G. Komen Breast Cancer
Foundation, and the Irma T. Hirschl Award (to N.C.).
Endocrine Reviews, February 2003, 24(1):48 –77
71
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2nd International Conference on the Female Reproductive Tract
Frauenchiemsee, Germany
May 30 –June 2, 2003
Organizing Committee: E. Winterhager, R. Gruemmer, K. Chwalisz, K. S. Korach
Deadline for registration and abstract: first of March
Website: www.uni-essen.de/anatomie/