PHYSIOLOGICAL REVIEWS
Vol. 80, No. 3, July 2000
Printed in U.S.A.
Molecular Analysis of the Sodium/Iodide Symporter:
Impact on Thyroid and Extrathyroid Pathophysiology
ANTONIO DE LA VIEJA, ORSOLYA DOHAN, ORLIE LEVY, AND NANCY CARRASCO
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York
I. Introduction
II. Characterization of the Sodium/Iodide Symporter Protein
A. Generation of anti-NIS antibodies
B. N-linked glycosylation of NIS: implications for the NIS secondary structure model
C. Extracellular orientation of the NH2 terminus of NIS
D. Several hydroxyl-containing amino acid residues in the transmembrane segment IX (Ser-353,
Thr-354, Ser-356, and Thr-357) are important for NIS function
E. Electrophysiological analysis of NIS: mechanism, stoichiometry, and specificity
F. The NIS inhibitor perchlorate is not translocated by NIS into the cell
III. Regulation of Sodium/Iodide Symporter Protein Expression
IV. Transcriptional Regulation of the Sodium/Iodide Symporter
A. Thyroid-specific transcription factors
B. Transcriptional regulation of Tg and TPO
C. Transcriptional regulation of TSHr
D. Analysis of the human NIS promoter
E. Analysis of the rat NIS promoter
V. Expression of Sodium/Iodide Symporter in Nonthyroid Tissues
A. NIS in salivary glands, stomach, mammary gland, and other tissues
B. Identification of mammary gland NIS and gastric NIS
C. Regulation of mammary gland NIS
VI. The Sodium/Iodide Symporter in Autoimmune Thyroid Disease
VII. The Sodium/Iodide Symporter and Cancer
VIII. Congenital Iodide Transport Defect Due to Sodium/Iodide Symporter Mutations
IX. Concluding Remarks
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De La Vieja, Antonio, Orsolya Dohan, Orlie Levy, and Nancy Carrasco. Molecular Analysis of the Sodium/
Iodide Symporter: Impact on Thyroid and Extrathyroid Pathophysiology. Physiol Rev 80: 1083–1105, 2000.—The
Na⫹/I⫺ symporter (NIS) is an intrinsic membrane protein that mediates the active transport of iodide into the thyroid
and other tissues, such as salivary glands, gastric mucosa, and lactating mammary gland. NIS plays key roles in
thyroid pathophysiology as the route by which iodide reaches the gland for thyroid hormone biosynthesis and as a
means for diagnostic scintigraphic imaging and for radioiodide therapy in hyperthyroidism and thyroid cancer. The
molecular characterization of NIS started with the 1996 isolation of a cDNA encoding rat NIS and has since
continued at a rapid pace. Anti-NIS antibodies have been prepared and used to study NIS topology and its secondary
structure. 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 thyrotropin and I⫺ has been analyzed, the
regulation of NIS transcription has been studied, spontaneous NIS mutations have been identified as causes of
congenital iodide transport defect resulting in hypothyroidism, the roles of NIS in thyroid cancer and thyroid
autoimmune disease have been examined, and the expression and regulation of NIS in extrathyroidal tissues have
been investigated. In gene therapy experiments, the rat NIS gene has been transduced into various types of human
cells, which then exhibited active iodide transport and became susceptible to destruction with radioiodide. The
continued molecular analysis of NIS clearly holds the potential of an even greater impact on a wide spectrum of
fields, ranging from structure/function of transport proteins to the diagnosis and treatment of cancer, both in the
thyroid and beyond.
www.physrev.physiology.org
0031-9333/00 $15.00 Copyright © 2000 the American Physiological Society
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I. INTRODUCTION
The thyroid is a master endocrine gland that plays a
central role in the intermediary metabolism of virtually all
tissues and is of fundamental importance for the development of the central nervous system in the fetus and the
newborn. The widespread effects of the thyroid result
from the biosynthesis and secretion of two rather unique
hormones, triiodothyronine (T3) and thyroxine (or tetraiodothyronine) (T4), the only iodine-containing hormones in vertebrates. Iodide (I⫺) is an essential constituent of T3 and T4 so that both thyroid function as a whole
and its systemic ramifications depend on an adequate
supply of I⫺ to the gland. A remarkably efficient and
specialized system has evolved in the thyroid that ensures
that most of the ingested dietary I⫺ (the only source of I⫺)
is accumulated in the gland and thus made available for T3
and T4 biosynthesis. The significance of this 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 (24). I⫺ deficiency still often leads to
various degrees of impaired brain development, mostly in
populations of children living in poor regions (114). 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 solutions like this from being
implemented, at great human cost. Still, this situation
dramatizes not only the health value of I⫺ as a nutrient
and the consequences to society of its environmental
scarcity, but also underscores how difficult it would be
for people to stay euthyroid and healthy in the absence of
a specialized I⫺ transport mechanism in the thyroid.
The ability of thyroid follicular cells to concentrate
I⫺ was first reported as early as 1915 (68). The thyroid
gland was found to be capable of concentrating I⫺ by a
factor of 20 – 40 with respect to its concentration in the
plasma under physiological conditions. Hence, the existence of a thyroid I⫺ transporter was inferred, and some
of its properties were elucidated over the years (see Refs.
16, 42, 43, 61, 63, 98, 107 and 125 for reviews). Briefly, I⫺
accumulation in the thyroid has long been shown to be an
active transport process that occurs against the I⫺ electrochemical gradient, stimulated by thyrotropin (TSH),
and blocked by the well-known “classic” competitive inhibitors, the anions thiocyanate and perchlorate. Eventually, it was determined that the thyroid I⫺ transporter is a
Na⫹/I⫺ symporter (NIS) (6, 9, 45, 79, 122), i.e., an intrinsic
plasma membrane transport protein that couples the inward “downhill” translocation of Na⫹ to the inward “uphill” translocation of I⫺ (Fig. 1). The driving force for the
process is the inwardly directed Na⫹ gradient generated
Volume 80
by the Na⫹-K⫹-ATPase. 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
pharmacological doses of radioiodide to target and destroy hyperfunctioning thyroid tissue, such as in Graves’
disease or I⫺-transporting thyroid cancer and its metastases (23, 124). Therefore, the study of NIS is of great
relevance to thyroid pathophysiology. Nevertheless, no
molecular information on NIS was available until 1996,
when after a decades-long search by numerous investigators, a cDNA encoding rat NIS was finally isolated by
expression cloning in Xenopus laevis oocytes (21). This
development, a major breakthrough in the study of I⫺
transport processes and thyroid physiology, marked the
beginning of the molecular characterization of NIS.
On the basis of the cloned cDNA, rat NIS was determined to be a protein of 618 amino acids (relative molecular mass 65,196) (Fig. 2). The hydropathic profile and
initial secondary structure predictions (20, 21) of the
protein suggested an intrinsic membrane protein with 12
putative transmembrane segments. The NH2 terminus
was originally placed 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 ⬃70 amino
acids within which the only potential canonical cAMPdependent protein kinase A (PKA) phosphorylation sequence of the molecule was located (positions 549 –552).
Three potential Asn-glycosylation sites were identified in
the deduced amino acid sequence at positions 225, 485,
and 497. The first was located in a predicted intracellular
hydrophilic loop, while the last two were located in the
last hydrophilic loop, a segment predicted to be on the
extracellular face of the membrane. The length of the 12
transmembrane segments in this original model ranged
from 20 to 28 amino acid residues, except transmembrane
segment V, which contained 18 residues. Only three
charged residues were 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. Out of a total of eight Trp
residues found in the membrane, six were located near
the extremes of transmembrane segments. Four Leu residues (positions 199, 206, 213, and 220) appeared to comprise a putative leucine zipper motif in transmembrane
segment VI. This motif, which has been proposed to play
a role in the oligomerization of subunits in the membrane,
has been conserved in all cloned neurotransmitter transporters (20, 21). NIS is often regarded to be a member of
the Na⫹/glucose cotransporter (SGLT1) family of transporters. For a detailed review of this family of transporters, see Reference 113a.
The molecular characterization of NIS has proceeded at an astounding pace with a wide variety of
July 2000
MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
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FIG. 1. Schematic representation of the biosynthetic pathway of thyroid hormones triiodothyronine (T3) and
thyroxine (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, and the apical surface on the right. Circle, active
accumulation of I⫺, mediated by the Na⫹/I⫺symporter (NIS); triangle, Na⫹-K⫹-ATPase; square, thyrotropin (TSH)
receptor; diamond, adenylate cyclase; ellipse, G protein; cylinder, I⫺ efflux toward the colloid; TPO, thyroid peroxidase
(TPO)-catalyzed organification of I⫺; arrows, endocytosis of iodinated thyroglobulin (Tg), followed by phagolysosomal
hydrolysis of endocytosed iodinated Tg and secretion of both thyroid hormones.
approaches and techniques, leading to numerous reports in just the last 3 years (61, 63, 98, 107). This
review provides an overview of the most recent developments on the molecular analysis of NIS, among
which are the following: anti-NIS antibodies have been
prepared and used to study the topology of NIS and to
experimentally test the proposed NIS secondary model
so that some aspects of the above-described model
have been experimentally confirmed and others revised. The most recent revised secondary structure
model for NIS proposes 13 (Fig. 2B) rather than 12
transmembrane segments (Fig. 2A). The biogenesis and
posttranslational modifications of NIS have been examined; a thorough electrophysiological analysis of NIS
has been conducted, in which NIS specificity and stoichiometry were studied and a mechanistic model was
proposed; the cDNA encoding human NIS (hNIS) has
been isolated; the genomic organization of hNIS has
been elucidated (Fig. 7) and the hNIS gene has been
mapped to chromosome 19p; the regulation of NIS by
TSH and I⫺ has been analyzed; the regulation of NIS
transcription has been studied; spontaneous NIS mutations have been identified as causes of congenital hypothyroidism; and the roles of NIS in thyroid cancer
and thyroid autoimmune disease have been examined.
In addition, it has been shown that I⫺ transport in
at least some extrathyroidal tissues, such as breast and
gastric mucosa, is also mediated by NIS expressed in
these tissues, in which NIS is differently regulated and
subjected to distinct posttranslational modifications.
This notion effectively invalidates the previously held
view that NIS was a major thyroid-specific protein, like
thyroglobulin (Tg) and thyroid peroxidase (TPO), presumably not expressed in any other tissues. Gene therapy experiments have recently been reported in which
the rat NIS gene was transduced into human melanoma,
murine liver, murine colon, and human carcinoma cells,
all of which then exhibited active I⫺ transport and
became susceptible to destruction with radioiodide.
Therefore, it is now clearer than ever before that the
continued molecular analysis of NIS holds the potential
of an even greater impact on a wide spectrum of fields,
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FIG. 2. Original and current NIS secondary structure models. Top: original NIS secondary structure
model with 12 putative transmembrane segments
(21). Both the NH2 and COOH termini are proposed
to face intracellularly. Two putative N-linked glycosylation consensus sequences are indicated with
branched symbols at positions 485 and 497. The third
N-linked glycosylation sequence at position 225 is
located in the third predicted intracellular loop. Bottom: current NIS secondary structure mode with 13
putative transmembrane segments. Both the hydrophilic loop containing N225 and the NH2 terminus
face extracellularly (64). All three N-linked glycosylation consensus sequences are indicated at positions
225, 485, and 497. Amino acid residues 389 – 410 form
a new transmembrane segment (X) between segments 9 and 10 of the original model (see top panel).
ranging from structure/function of transport proteins to
the diagnosis and treatment of cancer, both in the
thyroid and beyond.
II. CHARACTERIZATION OF THE
SODIUM/IODIDE SYMPORTER PROTEIN
A. Generation of Anti-NIS Antibodies
A major development in the molecular characterization of NIS has been the generation of anti-NIS antibodies
(Ab). Levy et al. (62) generated a high-affinity [dissociation constant (Kd) ⬃100 pM] site-directed polyclonal antiNIS Ab against the last 16 amino acid residues of the
COOH terminus of the protein. The Ab immunoreacts
with a mature ⬃87-kDa polypeptide (i.e., NIS) and a partially glycosylated (⬃56 kDa) polypeptide in a line of
highly functional thyroid rat cells (FRTL-5 cells). Immunoreactivity is also observed in X. laevis oocytes and COS
cells expressing NIS, and it is competitively blocked by
the presence of excess synthetic peptide. This anti-COOHterminal NIS Ab was the first available tool to experimentally probe the NIS secondary structure model, and it was
used to confirm the model-predicted cytosolic-side location of the COOH terminus by indirect immunofluorescence experiments in permeabilized FRTL-5 cells (62).
Subsequently and independently, another site-directed Ab
against the same COOH-terminal segment of NIS was
generated by Paire et al. (81), which similarly immunore-
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MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
acts with an ⬃80- to 90-kDa glycosylated NIS protein from
FRTL-5 cells.
B. N-Linked Glycosylation of NIS: Implications for
the NIS Secondary Structure Model
Paire et al. (81) used their anti-NIS COOH Ab to
explore the regulation of NIS by TSH in FRTL-5 cells.
They observed that tunicamycin, an inhibitor of the synthesis of N-linked oligosaccharides, prevented both the
synthesis of mature NIS and the TSH-dependent reinduction of NIS activity in FRTL-5 cells. On this basis, they
suggested that N-linked glycosylation of NIS was essential
for NIS biosynthesis, correct folding, and stability. However, because tunicamycin inhibits the synthesis of Nlinked oligosaccharides and thus prevents N-linked glycosylation of all proteins in the cell, it is clear that the
observed effect of tunicamycin on NIS activity is not
necessarily due specifically to the lack of N-linked glycosylation of NIS. Levy et al. (64) have obtained conclusive
evidence showing that, contrary to the conclusion of
Paire et al., neither partial nor total lack of N-linked
glycosylation impairs activity, stability, or targeting of
NIS. Using site-directed mutagenesis, Levy et al. (64) substituted both separately and simultaneously the Asn residues (amino acids 225, 485, and 497, see Fig. 2) in all three
putative N-linked glycosylation consensus sequences of
NIS with Gln and assessed the effects of the mutations on
function, targeting, and stability of NIS in COS cells. All
mutants were active and displayed 50 –100% of wild-type
NIS activity, including the completely nonglycosylated
triple mutant, which migrated as a ⬃50-kDa NIS polypeptide. They observed that the half-life of nonglycosylated
NIS was similar to wild-type NIS and that the Michaelis
constant (Km) value for I⫺ (⬃30 ␮M) in nonglycosylated
NIS was virtually identical to wild-type NIS. These findings demonstrate that, to a considerable extent, function,
targeting, and stability of NIS are present even in the total
absence of N-linked glycosylation (64). Therefore, a bacterial expression system, in which no N-linked glycosylation occurs, may be used to overproduce NIS for structural studies.
In their report of N-linked glycosylation of NIS, Levy
et al. (64) demonstrated that the putative N-linked glycosylation site at N225, which had originally been predicted
to face intracellularly (Fig. 2A), is indeed glycosylated.
Therefore, it is now clear that the hydrophilic loop that
contains this sequence faces the extracellular milieu
rather than the cytosol. They have proposed a 13-transmembrane segment model to be the most likely secondary
structure for NIS. In contrast to the original model, in
which the NH2 terminus was predicted to face the cytosol
based on the lack of a signal sequence in NIS, in the
current model both the NH2 terminus and the hydrophilic
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loop containing N225 are predicted to be on the extracellular side, and the COOH terminus facing the cytosol, as
confirmed previously (Fig. 2B).
C. Extracellular Orientation of the NH2 Terminus
of NIS
Levy et al. (64) have recently demonstrated unequivocally that the NH2 terminus faces the external milieu, as
proposed in the current model. This conclusion was
reached using two independent experimental approaches.
First, these authors introduced a FLAG (MDYKDDDDK)
epitope into the NH2 terminus. COS cells transfected with
FLAG-containing NIS displayed undistinguishable I⫺ uptake accumulation from COS cells transfected with wildtype 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 technique took advantage
of a previous observation that unglycosylated NIS is active. The N-linked glycosylation amino acid sequence
NNSS was introduced into the NH2 terminus of unglycosylated NIS (25). They observed glycosylation of NIS at
the NH2 terminus upon transfection of NNSS-containing
NIS into COS cells, thus proving that the NH2 terminus
faces the lumen of the endoplasmic reticulum during
biosynthesis and therefore faces the external milieu upon
reaching the plasma membrane (25).
In addition, utilizing the same strategy of N-linked
glycosylation scanning mutagenesis, De la Vieja et al. (25)
have demonstrated that the hydrophilic loop between
putative transmembrane segments VIII and IX faces the
external milieu (Fig. 2B). 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 outside Cys-less background, a mutant that retains total activity. NIS activity was modified by membrane-impermeable sulfhydryl reagents such as sodium
(2-sulfonatoethyl) methanethiosulfonate (MTSES) and
2-(trimethylamonium)ethyl methanethiosulfonate (MTSET), indicating the external localization of this residue.
In summary, 5 NIS loops (NH2 terminus, loops between
transmembrane segments IV and V, VI and VII, VIII and IX,
and XII and XIII) out of a total of 7 have experimentally
been confirmed to have the external disposition predicted
in the current 13-transmembrane segment secondary
structure model. Additional experiments are being carried
out to complete the topological analysis of NIS.
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D. Several Hydroxyl-Containing Amino Acid
Residues in the Transmembrane Segment IX
(Ser-353, Thr-354, Ser-356, and Thr-357) Are
Important for NIS Function
Levy et al. (65) have demonstrated that a hydroxyl
group at the ␤-carbon at position 354 (in the transmembrane segment IX) is essential for NIS function. 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 (39, 40, 55, 70). Patients with
this condition do not accumulate I⫺ in their thyroids,
often resulting in severe hypothyroidism. Seeking to characterize this transport defect at the molecular level, Levy
et al. (65) set out to determine whether T354P NIS is a
nonfunctional but stable polypeptide properly targeted to
the plasma membrane, or a fully or partially functional
protein that is retained in intracellular organelles as a
result of the mutation. For this purpose, Levy et al. (65)
generated T354P NIS by site-directed mutagenesis and
used their high-affinity anti-NIS Ab to monitor T354P expression in COS cells transfected with NIS cDNA. COS
cells transfected with T354P NIS cDNA were assayed for
I⫺ uptake activity and found to display no I⫺ accumulation. After demonstrating that T354P was properly targeted to the plasma membrane, these authors carried out
several additional amino acid substitutions at position 354
and determined that the lack of I⫺ transport activity is not
due to a structural change induced by proline, as had been
previously proposed (39), but rather to the absence of a
hydroxyl group at the ␤-carbon at position 354.
Significantly, the transmembrane segment IX, in
which Thr-354 is located, is where the highest incidence
of hydroxyl-containing amino acids is found in NIS (Fig.
2B). Hence, De La Vieja et al. (unpublished data) assessed
the role played by these other hydroxyl groups in NIS
function by replacing the corresponding amino acid residues with Ala and Pro. Substituting Ser-349, Thr-351, Ser358, and Thr-366 (Fig. 3) for similar amino acid residues
devoid of hydroxyl groups did not affect either NIS expression or activity. In contrast, 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. The
lack of function when Ala or Pro was present instead at
these positions was not due to an effect on expression or
trafficking, given that the mutant NIS proteins were
shown by confocal immunofluorescence to reach the
plasma membrane properly. Additional experiments are
being carried out to determine the mechanistic role of
these hydroxyl groups in NIS activity. Kinetic analyses are
also being performed to differentiate between kinetically
impaired and uncoupled NIS molecules.
FIG. 3. Amino acid sequence and ␣-helical wheel projection of the
transmembrane segment IX of NIS. A: amino acid sequence. Hydroxylcontaining residues are shown in bold. B: ␣-helical wheel projection.
Hydroxyl-containing residues are circled. Important residues for NIS
activity are shaded.
E. Electrophysiological Analysis of NIS:
Mechanism, Stoichiometry, and Specificity
Eskandari et al. (34) have examined the mechanism,
stoichiometry, and specificity of NIS by means of electrophysiological, tracer uptake, and electron microscopic
methods in X. laevis oocytes expressing NIS. These investigators obtained electrophysiological recordings using the two-microelectrode voltage-clamp technique.
They 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. Because 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⫹ 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⫹ (Fig. 4).
Eskandari et al. (34) observed also that in response
to step voltage changes, NIS exhibited current transients
that relaxed with a time constant of 8 –14 ms and that
pre-steady-state charge movements (integral of the current transients) versus voltage relations obeyed a Boltzmann distribution. These charge movements are attrib-
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MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
FIG. 4. Schematic representation of a NIS mechanistic model. As
shown in this scheme, first 1 Na⫹ binds to NIS, which in the absence of
substrate is able to cross the membrane via NIS in a Na⫹ uniport mode
(CNa⬘3 CNa⬙; C, carrier). Release of Na⫹ into the cytoplasm is followed
by the return of the empty binding site to complete the pathway (C⬙ 3
C⬘). The kinetic data suggest that Na⫹ binds to NIS before I⫺. In the
presence of I⫺, the complex CNa2I⬘ is formed which undergoes a conformational change to expose the bound I⫺ and 2 Na⫹ to the interior of
the cell (CNa2I⬘ 3 CNa2I⬙). Both Na⫹ and I⫺ are released into the
cytoplasmic compartment, and the empty carrier undergoes another
conformational change to expose the binding sites to the external solution again. 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 (C⬘3 C⬙). For more details, refer to
Reference 34.
uted to the conformational changes of the empty
transporter within the membrane electric field. These authors determined that the turnover rate of NIS is ⬃36 s⫺1
and reported that expression of NIS in oocytes led to a
⬃2.5-fold increase in the density of plasma membrane
protoplasmic face intramembrane particles, as ascertained by freeze-fracture electron microscopy. This is the
first direct electron microscopy visualization of ostensible
NIS molecules present in the oocyte plasma membrane.
Moreover, on the basis of their kinetic results, these investigators proposed an ordered simultaneous transport
mechanism in which Na⫹ binds to NIS before I⫺, i.e.,
whereas transport of both ions is simultaneous and binding is ordered and sequential. The combined data from
electrophysiological measurements and freeze-fracture
electron microscopy (9-nm-diameter NIS particles) provided in this report suggest that NIS may be multimeric in
its functional form (34).
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gesting that it is not transported (34). Yoshida et al. (130)
have reported, similarly, that perchlorate did not induce
an inward current in Chinese hamster ovary (CHO) cells
stably expressing NIS, as measured using the whole cell
patch-clamp technique. The most likely interpretation of
these observations is that perchlorate is not transported
by NIS, although the unlikely possibility that perchlorate
is translocated by NIS on a 1:1 Na⫹/ClO⫺
4 stoichiometry
cannot be ruled out. Therefore, perchlorate is a potent
inhibitor of NIS most probably acting as a blocker, not as
a substrate.
These results raise the question of whether or not
perchlorate is indeed translocated into thyroid cells expressing endogenous NIS or into cells other than oocytes
expressing transfected NIS, and this question has been
the subject of some controversy. In the acknowledgments
section of his recent extensive review on perchlorate and
the thyroid gland, Wolff (127) asserts that “perchlorate
accumulation in thyroid tissue has been repeatedly demonstrated” and suggests that the absence of perchlorate
transport by NIS in oocytes reported by Eskandari et al.
(34) may be due to differences between the oocyte system
and thyroid tissue. However, Yoshida et al. (131) thereafter showed that perchlorate elicits no change in the membrane current in the highly functional rat thyroid cell line
FRTL-5, as revealed by the whole cell patch-clamp technique, thus strongly suggesting that perchlorate is not
transported into FRLT-5 cells and supporting both their
previous observations in CHO cells (130) and the results
of Eskandari et al. (34) in oocytes.
With the consideration that the properties of NIS
expressed in oocytes are virtually indistinguishable from
those of endogenous NIS in thyroid cells, including
FRTL-5 cells, it appears that earlier experiments (42, 43,
F. The NIS Inhibitor Perchlorate Is Not
Translocated by NIS Into the Cell
Similar steady-state inward currents were generated
by a wide variety of anions in addition to I⫺ (including
⫺
⫺
⫺
⫺
⫺
⫺
⫺
ClO⫺
3 , SCN , SeCN , NO3 , Br , BF4 , IO4 , and BrO3 ),
indicating that these anions are also transported by NIS.
However, perchlorate (ClO⫺
4 ) (Fig. 5), the most widely
characterized inhibitor of thyroidal I⫺ uptake, was surprisingly found not to generate a current, strongly sug-
FIG. 5. Substrate selectivity of NIS. Inward currents induced by
various anions (500 ␮M) were recorded at a membrane potential of ⫺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 that did not induce a
⫺
2⫺
2⫺
detectable inward current were as follows: NO⫺
2 , HCO3 , SO3 , CO3 ,
4⫺
S2O2⫺
3 , P2O4 . Data are reported as means ⫾ SE (n ⫽ 3). [Adapted from
Eskandari et al. (34).]
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DE LA VIEJA, DOHAN, LEVY, AND CARRASCO
125) ostensibly showing that 36Cl-labeled perchlorate enters the cell may have been misinterpreted. Because
36
[36Cl]chlorate (ClO⫺
Cl-labeled by-product of the
3 ) is a
reaction employed to chemically synthesize [36Cl]perchlorate (ClO⫺
4 ) for these uptake studies, it seems likely
that [36Cl]chlorate, rather than perchlorate, accounts for
the presence of label in the cytosol of thyrocytes, given
that chlorate is readily translocated via NIS into the cell
(34). Current electrophysiological data, in conclusion,
strongly indicate that perchlorate is not translocated via
NIS into the cell.
III. REGULATION OF SODIUM/IODIDE
SYMPORTER PROTEIN EXPRESSION
TSH is the primary hormonal regulator of thyroid
function overall and has long been known to stimulate
thyroidal I⫺ accumulation. TSH is a glycoprotein of
⬃30,000 Da, biosynthesized in the adenohypophysis by
basophilic cells known as thyrotropes. The release of TSH
from the pituitary is stimulated by thyrotropin-releasing
hormone (TRH) from the hypothalamus and inhibited
through a negative-feedback mechanism by the thyroid
hormones T3 and T4. Most actions of TSH take place
through activation of adenylate cyclase via the GTP binding protein G␣ (117). This cascade of events is initiated by
the interaction of TSH with its receptor [i.e., TSH receptor
(TSHr)] on the basolateral membrane of the follicular
cells (Fig. 1). Early observations made before the isolation of the NIS cDNA suggested that TSH stimulation of I⫺
accumulation results, at least in part, from the cAMPmediated increased biosynthesis of NIS (51, 122). Using
high affinity anti-NIS Ab, Levy et al. (62) demonstrated in
rats that NIS protein expression is upregulated by TSH in
vivo. They prepared thyroid membrane fractions from
control, propylthiouracil (PTU)-treated, iodine-deficient,
and hypophysectomized rats, the latter with or without
subsequent injection of TSH. Membrane fractions were
then subjected to immunoblot analysis with anti-NIS Ab.
They observed NIS upregulation in rats with increased
TSH circulating levels caused either by PTU treatment
(which inhibits I⫺ organification) or an I⫺-deficient diet.
Conversely, NIS protein expression was decreased in hypophysectomized rats, which exhibit markedly lower TSH
levels. A single injection of TSH to hypophysectomized
rats reinduced the expression of NIS protein back to basal
levels. Consistent with these findings is a later observation by Uyttersprot et al. (115) that the expression of NIS
mRNA in dog thyroid (⬃3.9 kb) is dramatically upregulated by goitrogenic treatment (i.e., PTU treatment, which
leads to elevated TSH circulating levels in vivo).
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. As early as 1944, Morton
Volume 80
et al. (75) reported that the biosynthesis of thyroid hormones by sheep thyroid slices was inhibited by high doses
of I⫺. Wolff and Chaikoff reported in 1948 (128) that
organic binding of iodide in the rat thyroid was blocked
when I⫺ thyroid levels reached a critical high threshold, a
phenomenon known as the acute Wolff-Chaikoff effect.
These researchers observed further that ⬃2 days later, in
the presence of continued high plasma I⫺ concentrations,
an “escape” or adaptation from the acute effect is observed so that the level of organification of I⫺ is restored
and normal hormone biosynthesis resumes (129).
Whereas the mechanism responsible for the acute WolffChaikoff effect has yet to be fully elucidated, it has been
proposed to be the result of organic iodocompounds acting as mediators. The iodolipid ␣-iodohexadecanal has
been suggested to be one such mediator, on account of its
ability to inhibit NADPH oxidase, TPO, and TSH-induced
cAMP formation in the thyroid (28). The less studied
mechanism for the escape from the acute Wolff-Chaikoff
effect has been proposed by Braverman and Ingbar (12) to
be due to a decrease in I⫺ transport, which would presumably lead to sufficiently low intracellular I⫺ concentrations to remove inhibition of I⫺ organification.
As in the case of NIS regulation by TSH, the regulatory role played by I⫺ on NIS function was explored at the
molecular level only after the cDNA that encodes NIS was
isolated. In vivo studies have shown that I⫺ inhibits the
expression of both TPO and NIS mRNA in dog thyroid
(115), a finding consistent with the Wolff-Chaikoff effect.
More recently, Eng et al. (33) measured the levels of NIS
mRNA and NIS protein in response to both chronic and
acute I⫺ excess in rats in vivo, thus showing, specifically,
that the decrease of I⫺ transport observed during the
escape from the Wolff-Chaikoff effect is due to a decrease
in NIS expression by a mechanism that is at least in part
transcriptional.
IV. TRANSCRIPTIONAL REGULATION OF THE
SODIUM/IODIDE SYMPORTER
A. Thyroid-Specific Transcription Factors
The transcriptional regulation of three other genes of
major significance in thyroid physiology had been analyzed, namely, the Tg, TPO, and TSHr genes. Initially, all
three gene products were proposed to be thyroid specific,
i.e., to be expressed exclusively in the thyroid. However,
TSHr expression and activity have recently been demonstrated in adipocytes (100), leaving Tg and TPO as the
only thyroid-specific proteins.
Three different transcription factors have been implicated in thyroid-specific gene transcription (22, 73): 1)
thyroid transcription factor (TTF)-1, a homeodomain
(HD)-containing protein present in the developing thy-
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MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
1091
roid, lung, forebrain, and pituitary and in the adult thyroid
and lung; 2) TTF-2, a forkhead protein detected in developing thyroid and anterior pituitary and in the adult thyroid; and 3) Pax8, a nuclear protein member of the murine
family of paired-domain (PD) containing genes, present in
the developing thyroid, kidney, and midbrain boundary
and in the adult thyroid and kidney. Specific combinations
of these factors have been proposed to regulate transcription of the thyroid-specific proteins Tg and TPO and also
of the TSHr.
(95) have recently shown that both TTF-2 and HNF-3␤,
another forkhead transcription factor also present in the
thyroid, bind to the same site and thus participate in the
regulation of the TPO but not the Tg promoter. In addition, upstream enhancers have also been identified in
both promoters. Three TTF-1 binding sites for the bovine
Tg promoter are present 2 kb upstream. Two TTF-1 and
one Pax8 binding site, recently reported (35), are found in
a 230-bp enhancer located 5.5 kb upstream of the TPO
initiation codon (22).
B. Transcriptional Regulation of Tg and TPO
C. Transcriptional Regulation of the TSHr
The Tg and TPO minimal promoter structures are
very similar to each other but different from the TSHr
promoter (22) (Fig. 6). The Tg and TPO promoters from
different species exhibit three binding sites for TTF-1 (A,
B, and C), one binding site for TTF-2, and one for Pax8. All
of these sites are localized 5⬘-upstream between ⫺170 and
⫹1 bp (A in the ATG initiation codon) and both contain a
TATA box. The TTF-1 site C and Pax8 site overlap, suggesting that this overlapping site area may play an important role in thyroid-specific transcription. TTF-2 plays an
important role in the modulation of the Tg and TPO genes
by insulin and insulin-like growth factor I, but this action
is more evident for the TPO than for the Tg promoter (8,
80, 94). Both promoters have been shown to have binding
sites for different ubiquitous transcription factors,
namely, ubiquitous factor A (UFA) in the Tg promoter and
ubiquitous factor B (UFB) in the TPO promoter. Sato et al.
The TSHr promoter is very different from the Tg and
TPO promoters (Fig. 6). The TSHr minimal promoter
region is localized in the 5⬘-upstream region between
⫺220 and ⫺39 bp and shows thyroid-specific expression
and TSH/cAMP regulation. Several binding regulatory elements have been proposed to play a role in the regulation of TSHr transcription. A canonical cAMP responsive
element (CRE) (⫺139 to ⫺131 bp) site that is autoregulated by TSH via cAMP has been shown to first increase
and then decrease the expression of TSHr as a function of
time (93). One TTF-1 binding site is present at ⫺189 to
⫺175 bp; binding of phosphorylated TTF-1 to this site
produces a modest effect on the activation of transcription (77, 101). Single-strand DNA-binding proteins
(SSBP), whose binding sites overlap with the 5⬘-end of the
TTF-1 site, have been implicated in the regulation of the
TSHr (102). In contrast to the Tg and TPO promoters,
FIG. 6. Schematic representation of the structures of the Tg, TPO, TSH receptor (TSHr), and NIS promoters. Symbols
refer to binding sites mapped by DNase I footprinting. Numbers indicate the distances from the corresponding
transcriptional start sites. For specific details, see Reference 22.
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DE LA VIEJA, DOHAN, LEVY, AND CARRASCO
neither TTF-2 nor Pax8 binding sites have been identified
in the TSHr promoter. The TSHr transcript has also been
found in retro-orbital fibroblasts and in adipose tissue (32,
60). Rat adipose tissue expresses TSHr mRNA levels similar to those found in the thyroid (100). The promoter
regulation is different in adipocytes from thyroid cells,
but both display multiple start sites and important regulation by CREB (99).
TPO and Tg expression are both upregulated by TSH
(41, 116) but by different regulatory mechanisms. TPO
and TSHr regulation takes place faster than Tg regulation.
Although Tg transcriptional activation kinetics depend on
the experimental model used, i.e., it is rapid (⬃1 h) in
thyroid slides and slow (⬃8 h) in primary cultures, TPO
induction is rapid in both experimental models (41). All
three genes are controlled in the thyroid at the transcriptional level mainly by a cAMP-dependent mechanism,
whose intracellular level is elevated by TSH/forskolin.
However, only the TSHr gene has been shown to be
regulated by cAMP through a CRE sequence. The exact
mechanism by which cAMP regulates Tg and TPO is still
an open question.
D. Analysis of the Human NIS Promoter
It has long been established that TSH stimulates NIS
activity via the cAMP pathway (62, 81, 122) and, more
recently, that it upregulates NIS mRNA levels (53). However, to fully understand these mechanisms, it is necessary to study the transcriptional regulation of NIS. The
human NIS promoter has been sequenced by three different groups (10, 90, 118). Venkataraman et al. (118) isolated a 1.2-kb fragment of the 5⬘-flanking region of the
hNIS gene. Significantly, they characterized the promoter
in a human thyroid cell line, KAT-50, and found thyroidspecific expression between ⫺1,044 and ⫺336 bp. Some
putative transcription factor binding sites, both thyroid
and nonthyroid, are localized in this region (1.2 kb) but
were not analyzed. It is noteworthy that this list includes
the putative TTF-2 and Pax8 binding sites, both of which
are found within the 2-kb portion of the 5⬘-flanking region
in the rat promoter (see below). Ryu et al. (90) isolated 2
kb of the 5⬘-flanking region from the hNIS promoter. They
mapped the start site to ⫺375 bp and delimited the minimal promoter between ⫺478 and ⫺389 bp. A 90-bp fragment exhibited a high identity (73%) with the minimal
rNIS promoter. These investigators reported some mismatches with the previously reported sequence. However,
this group did not find thyroid-specific regulatory elements. Finally, Behr et al. (10) isolated 1.6 kb of the
5⬘-flanking region of the hNIS gene. Like Ryu et al. (90),
Behr et al. (10) also found strong promoter activity in
thyroid and nonthyroid cells within the minimal promoter. Behr et al. (10) suggested that the nonspecificity
Volume 80
could result from the presence of an upstream tissuespecific enhancer or from the influence of chromatin
structure and/or methylation, as there are numerous CpG
dinucleotides.
E. Analysis of the Rat NIS Promoter
Tong et al. (112) isolated a 16.4-kb fragment of
genomic rNIS DNA. They localized the start site at ⫺98 bp
and one TATA box between ⫺124 and ⫺118 (AATAAAT),
with a minimal promoter localized between ⫺199 and
⫺110 bp. Surprisingly, however, they concluded that 8 kb
of the 5⬘-flanking region of the NIS gene is not sufficient to
confer thyroid-specific transcription. Neither TTF-2 nor
Pax8 putative binding sites were localized within 2 kb
upstream of the initiation codon. However, the conclusion of Tong et al. (112) proved incorrect, because Endo
et al. (30) localized a TTF-1 binding site between ⫺245
and ⫺230 bp, in the proximal rNIS promoter (2 kb), that
confers thyroid-specific transcription but only exerts a
modest effect.
The same group (76) subsequently identified, in the
5⬘-flanking region between ⫺1,968 and ⫹1 bp, a novel TSHresponsive element (TRE) between ⫺420 and ⫺385 bp upstream of the TTF-1 site in the rNIS promoter, that upregulated two- to threefold NIS expression. The TSH effect is
cAMP mediated and thyroid specific. They showed that the
protein that binds this site is different from TTF-1, TTF-2,
Pax8, or other known transcription factors, and the researchers named the putative binding protein in the TRE site
NIS TSH-responsive factor 1 (NTF-1). However, because the
TSH upregulation of NIS via this TRE site is lower than the
regulation that the same group reported previously (⬃6fold) (53), they have suggested that other transcription binding sequences may be present upstream from the 1,968-bp
region that they studied.
A thorough characterization of the upstream enhancer of the rNIS gene has recently been reported by
Ohno et al. (78). These investigators showed that the rNIS
regulatory region contains a nonthyroid-specific promoter
between ⫺564 and ⫺2 bp and an enhancer located between ⫺2,264 and ⫺2,495 bp that recapitulates the most
relevant aspects of NIS regulation. This rNIS enhancer
mediates thyroid-specific gene expression by the interaction of Pax8 with a novel cAMP-dependent pathway. The
NIS upstream enhancer (NUE) stimulates transcription in
a thyroid-specific and cAMP-dependent manner. NUE
contains the following: two Pax8 binding sites (PA and
PB); two TTF-1 binding sites (TA and TB) that have no
effect on rNIS transcription; and a degenerate CRE sequence (5⬘-TGACGCA-3⬘), which is important for NUE
transcriptional activity. Interestingly, the same degenerate CRE sequence has been implicated in tissue-specific
cAMP response of other promoters, such as the dopamine
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MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
␤-hydroxylase (111), prohormone convertase 1 (47), and
proenkephalin genes (19). In NUE, both Pax8 and the
unidentified CRE-like binding factor act synergistically to
obtain full TSH/cAMP-dependent transcription. However,
this enhancer is also able to mediate cAMP-dependent
transcription by a novel PKA-independent mechanism
(78).
The present picture of the NIS promoter shows two
important regions (Fig. 6): 1) a proximal promoter, still
questionably thyroid specific, reported to be TSH/cAMP
regulated by a TRE binding sequence, mediated by a new
NTF-1 protein (76). This upregulation, however, is lower
than the TSH-induced mRNA expression reported by the
same group (53). 2) The NIS promoter also has an upstream enhancer that is thyroid specific, with Pax8 and
CRE-like binding factors. It seems likely that both regions
in the promoter act synergistically to achieve full highlevel transcription.
Transcriptional regulation of the Tg, TPO, and TSHr
genes by TSH/forskolin is mediated by cAMP. However,
no CRE sequences have been identified for any of these,
except the TSHr gene. Kambe et al. (49) have reported
that redox regulation of TTF-1 and Pax8 was involved in
the TSH-increased DNA-binding activities of these two
factors. However, no clear evidence has been provided on
the control exerted by TSH/cAMP. Thus the novel PKAindependent mechanism reported by Ohno et al. (78) is
highly significant, because it establishes a direct relationship between thyroid-specific transcription factors and
the TSH/cAMP regulation in rNIS. This picture clearly
separates rNIS gene regulation from that of true thyroidrestricted genes (Tg and TPO) and also from TSHr, a
notion consistent with the fact that NIS is not exclusively
expressed in the thyroid gland. It is clearly of much
interest to investigate the transcriptional regulation of
NIS in extrathyroidal tissues. These studies will reveal
mechanisms of different tissue-specific regulation of the
same gene and may provide insights into possible applications of NIS in the diagnosis and treatment of both
thyroid and nonthyroid cancer.
V. EXPRESSION OF SODIUM/IODIDE
SYMPORTER IN NONTHYROID TISSUES
The topic of I⫺ transport systems outside the thyroid
was extensively reviewed in 1961 by Brown-Grant (13).
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, but they also display important differences:
1) nonthyroid I⫺ transporting tissues do not have the
1093
ability to organify accumulated I⫺; therefore, they behave
like PTU-treated thyroid tissue; 2) TSH exerts no regulatory influence on nonthyroid I⫺ accumulation; and 3) at
least salivary glands and gastric mucosa concentrate thiocyanate, unlike the thyroid where it is metabolized (oxidized). 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 (13,
16, 42, 125). Moreover, thyroidal and extrathyroidal I⫺
accumulation generate I⫺ concentration gradients of similar magnitude (⬃20- to 40-fold under steady-state conditions). Hence, the isolation and characterization of the
NIS cDNA from rat thyroid (21) made it possible to examine NIS expression in nonthyroid tissues, leading to the
conclusion that I⫺ transport in most (and probably all)
extrathyroid tissues in which it is present is also mediated
by NIS itself, like in the thyroid. However, NIS is clearly
regulated and processed differently in each tissue.
A. NIS in Salivary Glands, Stomach, Mammary
Gland, and Other Tissues
Spitzweg et al. (108) subjected several extrathyroidal
human tissues to Northern blot and RT-PCR analysis with
human thyroid NIS cDNA and detected the NIS transcript
by Northern blot predominatly in the parotid salivary
gland but not in the other tissues. With the use of RT-PCR,
hNIS gene expression was detectable in salivary glands,
pituitary gland, pancreas, testis, mammary gland, gastric
mucosa, prostate, and ovary but not in orbital fibroblasts,
colon, and nasopharyngeal mucosa. Kotani et al. (56),
who carried out Northern analyses with rat thyroid NIS
cDNA in extrathyroidal rat tissues, detected the NIS transcript primarily in the stomach. Subsequently, Spitzweg et
al. (108) reported the cloning of human NIS cDNA from
gastric mucosa, parotid, and mammary glands, all of
which exhibited full identity to thyroid hNIS cDNA. Consistent with these findings, immunohistochemical studies
by Jhiang et al. (48) and by Kotani et al. (56) detected NIS
protein in the acinar cells of human salivary glands and in
surface epithelial and parietal cells of the rat stomach,
respectively. Ajjan et al. (4) have reported variable
amounts of hNIS amplified product, as detected by RTPCR, in human stomach, salivary gland, and mammary
tissue, but not in the ovary, esophagus, colon, extraocular
fat, or skin. Surprisingly, they failed to detect NIS expression using Northern blot analysis in stomach, salivary
gland, intestinal fat, and multinodular goiter tissue samples.
It must be pointed out that the RT-PCR technique
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DE LA VIEJA, DOHAN, LEVY, AND CARRASCO
yields a large number of false positives due to the high
sensitivity of the RT-PCR technique (38). 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 or results obtained by
RT-PCR and even Northern analyses, and 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 with the consideration of the above results, NIS appears to be expressed and active in extrathyroidal tissues
previously known to exhibit NIS activity, such as salivary
glands and gastric mucosa. The significance of the detection of the NIS-amplified product by RT-PCR in other
human and rat tissues still awaits further characterization.
B. Identification of Mammary Gland NIS and
Gastric NIS
Levy et al. (62) performed immunoblot analyses to
assess whether a high-affinity antithyroid NIS Ab would
react with a mammary gland membrane protein (111a).
They observed immunoreactivity against a single broad
⬃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 mammary gland NIS (mg-NIS). These authors 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. They treated membrane proteins
from thyroid and lactating mammary gland with N-glycosidase F, an enzyme that removes N-linked carbohydrates, and probed membranes with anti-NIS Ab. Under
these conditions, anti-NIS Ab recognized a ⬃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 ⬃75- and ⬃50kDa immunoreactive polypeptides detected in lactating
mammary gland correspond to glycosylated and nonglycosylated mg-NIS, respectively. Moreover, Tazebay et al.
(111a) also observed immunoreactivity of anti-NIS Ab
with a ⬃100-kDa gastric polypeptide, which upon deglycosylation migrated, too, at ⬃50 kDa. In all likelihood,
these polypeptides correspond, respectively, to glycosylated and nonglycosylated gastric NIS (g-NIS). CNBr
treatment of rat thyroid NIS, mg-NIS, and g-NIS proteins
yielded the same peptide map in each case (Levy et al.,
Volume 80
Ref. 62), a finding consistent with the identity among
human thyroid NIS, mg-NIS, and g-NIS predicted by the
cloning of human NIS cDNA from gastric mucosa and
mammary glands by Spitzweg et al. (108).
C. Regulation of Mammary Gland NIS
Subsequently, Tazebay et al. (111a) investigated
whether a correlation existed between I⫺ accumulation
activity and mg-NIS expression in the various physiological stages of the mammary gland. They found that mgNIS was absent in nubile and pregnant mammary gland,
but clearly present in lactating mammary gland. To ascertain whether weaning had an effect on mg-NIS expression, mother rats were removed from their litters. Twentyfour hours after weaning, mg-NIS expression was
significantly decreased, and after 48 h, it was barely detectable. Furthermore, mg-NIS expression was reversible
upon reestablishment of nursing. These results indicate
that mg-NIS expression is clearly upregulated during active nursing, rapidly downregulated upon cessation of it,
and exquisitely regulated in a reversible manner by suckling.
To assess the issue of tissue distribution of I⫺ transport activity, Tazebay et al. (111a) carried out in vivo
scintigraphic imaging of 131I⫺ (or the substitute isotope
99m
TcO⫺
4 ) tissue distribution in rats. In nonlactating rats,
the isotope was concentrated in the stomach and later in
the thyroid, whereas in lactating rats it was evident in the
stomach and soon thereafter in all pairs of mammary
glands. Concentration of the isotope in all these tissues
was inhibited by perchlorate.
VI. THE SODIUM/IODIDE SYMPORTER
IN AUTOIMMUNE THYROID DISEASE
An important aspect of the wide pathophysiological
impact of Tg, TPO, and TSHr is the fact that autoAb
against all three proteins have been demonstrated in patients suffering from autoimmune thyroid disease (AITD).
AITD is significantly more prevalent in women; it affects
⬃2% of the female population but only ⬃0.2% of males
(121). The pathogenesis of AITD across the spectrum
from Graves’ disease to Hashimoto’s thyroiditis has been
suggested to involve a deficiency in the number and/or
function of antigen-specific and nonspecific population of
T lymphocytes (37). As is the case in all autoimmune
disorders, multiple factors are believed to play a role in
the development of AITD, including genetic predisposition, environmental factors, and a state of dysregulation
of the immune system (37). In the presence of AITD, the
thyroid is infiltrated with mononuclear cells, including
activated T and B plasma cells and macrophages, thus
creating a local cytokine milieu (120). A distinctive fea-
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MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
ture of AITD is the expression of HLA class II molecules,
induced by the T cell secreted interferon (IFN)-␥, on the
follicular epithelial cells, which are normally class II negative. Another characteristic feature of AITD is the presence in patients’ sera of autoAb that are directed against
specific thyroid antigens (Tg, TPO, and TSHr) (121). Iodine is an important susceptibility and modulating factor
in the development of thyroid autoimmunity. High levels
of iodine can cause thyroid damage and thyroiditis, particularly against the background of a previously low intake of iodine. Significantly, the incidence of AITD is
higher in areas of iodine sufficiency than in areas of iodine
deficiency (37).
Given that thyroid-associated proteins such as Tg,
TPO, and TSHr are known targets for autoAb in AITD, the
molecular identification of NIS has made it possible for
the first time to experimentally assess whether NIS is also
a target autoantigen, and whether any anti-NIS autoAb (if
present) have an effect on NIS function. In addition, it is
also possible that NIS is one of the modulators of the local
autoimmune processes, conceivably regulating the individual I⫺ supply to the follicular cell.
Even before the isolation of the NIS cDNA, Raspe et
al. (88) screened sera from 147 patients with AITD by
measuring the effect of the sera on I⫺ transport activity in
primary cultures of dog thyrocytes. They observed that
the serum from a 58-yr-old female patient suffering from
Hashimoto’s thyroiditis, autoimmune gastritis, and rheumatoid arthritis, with high autoAb titers against Tg, TPO,
and gastric parietal cells, completely blocked TSH-induced I⫺ uptake. Inhibition was specific, given that the
serum was still active at 1:1,000 dilution and did not
inhibit the activity of the Na⫹-K⫹-ATPase. Unfortunately,
no further experiments were performed because only a
limited amount of serum was available. Nevertheless, the
investigators still concluded that the serum contained
NIS-inhibiting autoAb. The low prevalence of the autoAb
positivity (1 of 147 sera) may be due to the purely functional nature of the assay, as only Ab against external
epitopes and with an I⫺ transport inhibitory effect would
be detected, as well as to the use of dog thyrocytes to
screen human sera.
Endo et al. (31) screened sera from patients with
AITD by recombinant NIS protein slot blotted onto nitrocellulose sheets. Twenty-two of 26 Graves’ disease sera
and 3 of 20 Hashimoto’s thyroiditis sera seemed to detect
the recombinant NIS protein, but the effect of these positive sera on I⫺ uptake was not tested. In a subsequent
study, the same group (29) concentrated exclusively on
Hashimoto’s thyroiditis samples. Four of 34 sera from
patients with Hashimoto’s thyroiditis immunoreacted
with a ⬃80-kDa polypeptide, as seen upon immunoblot
analysis of FRTL-5 cell membranes (which contain not
only NIS but also other thyroid antigens), and these same
four sera caused 14 – 62% inhibition of I⫺ accumulation in
1095
CHO cells stably expressing recombinant rat NIS. The
investigators observed also that some normal sera and
patients’ sera that did not immunoreact on immunoblots
nevertheless caused ⬃90% inhibition of NIS activity. Because this inhibitory effect was lost after dialyzing these
sera, the authors concluded that some sera contained a
dialyzable inhibitor, independent of the presence or absence of anti-NIS autoAb, and thus suggested that purified
IgG be used to accurately evaluate the putative autoAb
activity. The investigators also concluded that the presence of anti-NIS autoAb may play a role in the pathogenesis of Hashimoto’s thyroiditis and thus may modulate
thyroid function. All of these preliminary observations
call for further study and confirmation.
Morris et al. (74) synthesized 21 peptides corresponding to putative extracellular segments of rat NIS, based on
the first 12 transmembrane segment secondary structure
model proposed for rat NIS (21). Samples were analyzed
by ELISA using the synthetically made peptides. Because
of the high variability of background binding, the normal
range of binding was determined in the case of each
peptide by a pool of normal IgG, and thereafter the number of standard deviation units from the mean of the
control was calculated. Eighty-eight percent of Graves’ (n
⫽ 27) and 63% of Hashimoto (n ⫽ 27) serum samples
recognized at least one synthetic peptide. The most highly
recognized eight peptides were those corresponding to
the fourth, fifth, and sixth extracellular loops and the
intracellular COOH-terminal tail of the first secondary
structure model (Fig. 2A), which correspond, respectively, to the fourth and sixth intracellular and sixth extracellular loops, and the intracellular COOH-terminal tail
of the current 13 transmembrane segments model (Fig.
2B). In contrast, none of the control sera displayed any
immunoreactivity. The occurrence of Ab that recognized
intracellular epitopes was explained by the investigators
as a result of exposure of these internal sequences to the
thyroiditis-induced follicular cell damage. Unfortunately,
there were no data regarding recognition of the entire NIS
molecule by these antisera.
Ajjan et al. (3) established a CHO cell line stably
expressing human NIS 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 IgG)
but none of the controls inhibited I⫺ uptake. The autoAb
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.
These experimental data suggest that NIS might be
an autoantigen in AITD and that autoAb are generated
1096
DE LA VIEJA, DOHAN, LEVY, AND CARRASCO
against it. It is important to continue the analysis of this
topic utilizing experimental systems free of other thyroid
autoantigens, and to investigate the presence of anti-NIS
autoAb against both linear and conformational epitopes,
that are no longer present after SDS-PAGE electrophoresis. Clearly, a wide range of experimental approaches will
be necessary to unequivocally determine the existence,
real prevalence, functional effects, and pathological significance of autoAb against NIS. Furthermore, another
interesting aspect to consider is the role of certain cytokines in NIS function, because recent reports indicate that
tumor necrosis factor (TNF)-␣ and, to lesser extent, interleukin (IL)-1␣ inhibit both basal and TSH-induced NIS
expression (5). High concentrations of IFN-␥ also downregulate TSH-stimulated NIS mRNA expression. Consequently, IL-1␣, TNF-␣, and IFN-␥ all inhibit I⫺ uptake in
FRTL-5 cells. Caturegli et al. (17) have recently generated
transgenic mice that produce IFN-␥ in the thyroid. The
presence of this cytokine in the thyroid had no effect on
Tg expression. In contrast, the expression of both TPO
and TSHr was slightly increased, whereas NIS expression
was significantly decreased. Consistently, I⫺ accumulation was severely reduced in these animals (17). Further
investigation on the effect of cytokines on I⫺ uptake may
reveal some correlations between I⫺ supply and the
pathogenesis of the autoimmune process.
VII. THE SODIUM/IODIDE SYMPORTER
AND CANCER
Although cancer of the thyroid is a relatively infrequent condition in the United States (0.6% of all cancers in
men and 1.6% in women, Ref. 104), it has a considerably
higher impact on endocrinological practice than would be
expected solely on the basis of its incidence. This is so
because the possible existence of thyroid cancer must be
ruled out whenever a thyroid nodule is detected. There is
a high estimated incidence of solitary palpable nodules in
United States adults (⬃2– 4%) (89). The degree of accumulation of I⫺, as revealed by scans of the gland, is used
as an aid in the differential diagnosis of thyroid nodules.
Thyroid nodules that accumulate I⫺ equally or more efficiently than the normal surrounding tissue are generally
benign, whereas most thyroid cancers display markedly
reduced I⫺ accumulation relative to healthy tissue. On the
other hand, carcinomas behaving as hyperfunctioning hot
nodules are very rare. Thus radioactive I⫺ plays a major
diagnostic and therapeutic role in the management of
differentiated thyroid carcinoma. The main morphological types of differentiated thyroid cancer are the papillary
carcinoma (75– 85% of all thyroid cancers) and the follicular carcinoma (10 –20%). In most instances, both types
are well-differentiated tumors that originate in the thyroid
follicular cells (89). Well-differentiated thyroid carci-
Volume 80
noma, either follicular or papillary, is one of the most
curable cancers; the overall survival rate at 10 years for
middle-aged adults with thyroid carcinoma is ⬃80 –90%
(97). The reduced I⫺ accumulation detected in the majority of thyroid cancers suggests that malignant transformation of these cells may have an effect on the NIS molecule.
Thus the continued characterization of NIS may shed light
on I⫺ transport changes observed in thyroid cancer.
The treatment of differentiated thyroid carcinoma is
total or near-total thyroidectomy followed by 131I ablation. Radioiodide ablation destroys occult microscopic
carcinomas and also any remaining normal thyroid tissue,
permitting postablative 131I total body scanning search for
possible persistent carcinoma (119). The tools that allow
the sensitive and specific detection and subsequent treatment of differentiated thyroid carcinoma are the uptake
(mediated by NIS) and organification (mediated by TPO)
of I⫺, both TSH-regulated processes. The tumorigenic role
of radioidine in the thyroid, transported also by NIS into
the follicular cell, is debated, but the increased incidence
of papillary thyroid carcinomas in children (100 times
higher than in nonexposed children) after nuclear testing
and nuclear accidents suggests that radioactive isotopes
of iodine have a direct tumorigenic effect on the thyroid
(97).
To understand the involvement of NIS in the observed patterns of I⫺ transport in thyroid carcinomas with
respect to healthy thyroid tissue, several groups have
looked for a possible correlation between the lower or
absent radioiodine uptake activity in thyroid carcinomas
and the expression of NIS mRNA or protein, using Northern blot analysis, RT-PCR, or immunological analyses.
Smanik et al. (106) observed, by Northern blot analysis,
lower expression of hNIS in two papillary, one follicular,
and one anaplastic carcinoma. Upon further analysis of
additional papillary carcinomas by RT-PCR, a more sensitive technique, they found that different papillary cancers expressed hNIS at variable levels.
Considering the limitations of RT-PCR as a quantitative method, Arturi et al. (7) were able to evaluate only the
presence or absence of the NIS transcript in a series of
malignant and benign thyroid tumors. They first analyzed
by RT-PCR 26 primary thyroid carcinomas (19 papillary, 5
follicular, and 2 anaplastic), and subsequently a new series of 15 follicular adenomas (11 cold and 4 hot as
classified by thyroid scintigraphy), and found loss of expression of NIS in 5 of 19 papillary cancers, 1 of 5 follicular cancers, and none detected in anaplastic tissues. All
the (benign) follicular adenomas except one expressed
NIS. According to these authors, the fact that even one
benign follicular adenoma failed to express NIS means
that the absence of the NIS mRNA signal may not be
restricted to the malignant phenotype. In all the studied
tumors, except anaplastic, both Tg and TPO transcripts
were expressed.
July 2000
MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
Caillou et al. (14) conducted an immunohistochemical analysis of NIS protein expression in thyroid pathological tissues, including six benign adenomas, five follicular, and nine papillary carcinomas. No immunostaining
for hNIS was detected in any of the six benign adenomas
that appeared to be hypofunctioning on thyroid scintigraphy. hNIS expression was lower or absent in the papillary
and follicular well-differentiated carcinomas, but more
NIS was present in these than in the poorly differentiated
follicular carcinomas. Consistent with these results,
Jhiang et al. (48) detected no hNIS by immunohistochemical staining in any of the three papillary or the single
follicular carcinoma that they studied. In contrast, Saito et
al. (92) found increased NIS expression in papillary carcinomas. They investigated 31 papillary carcinomas by
either Northern blot or immunological analysis. Based on
their observations of higher NIS expression in ⬃50% of
the analyzed carcinomas compared with nonmalignant
thyroid tissue, the investigators concluded that despite
the high amounts of NIS protein present, well-differentiated thyroid carcinomas do not accumulate radioiodide,
suggesting that the activity of NIS is impaired or absent as
a result of different posttranslational modifications, trafficking alterations, or other unknown modulatory factors.
In cancerous follicular cells positive for hNIS, the localization of the protein was described as basolateral and
intracellular (92).
In light of the data summarized above, one may conclude that hNIS expression may be either increased, decreased, or absent in well-differentiated thyroid cancer,
even though I⫺ transport activity is consistently decreased in the majority of tumors. Therefore, the explanation for the decreased I⫺ uptake in well-differentiated
thyroid carcinomas lies not simply in lower NIS expression, but more likely involves a more complex combination of regulatory changes ultimately affecting NIS expression, targeting, and/or activation. To date, there are
still no data concerning the regulation of expression,
posttranslational modifications, targeting to the plasma
membrane, or other factors regulating NIS in cancerous
follicular cells. Thus there clearly is a pressing need to
investigate a much larger number of thyroid cancer specimens by more quantitative techniques to better understand the expression of hNIS both at the transcript and
protein levels, to elucidate the cellular localization of NIS,
and to be able to compare such findings to the clinical
behavior of the carcinomas.
Some in vitro experiments concerning 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. Schimura et al. (96) 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. (67)
1097
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. If positive results were
obtained in subsequent in vivo experiments, this gene
therapy approach would undoubtedly be one of the most
promising developments concerning the possible uses of
the molecular characterization of NIS in the diagnosis and
treatment of cancer.
VIII. CONGENITAL IODIDE TRANSPORT
DEFECT DUE TO SODIUM/IODIDE
SYMPORTER MUTATIONS
Congenital lack of I⫺ transport is a rather uncommon
thyroid condition that has been defined as an I⫺ transport
defect (ITD). The general clinical picture consists of hypothyroidism (which can be normalized in some cases
with high I⫺ supplementation or L-T4 substitutive therapy), goiter, low thyroid I⫺ uptake (as determined by
scintigraphy), and low saliva-to-plasma I⫺ ratio (109, 126).
Even though congenital hypothyroidism is an infrequent
disease (incidence 1:3,000 –1:4,000 in neonates) (113), 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 (2,
11), Tg (60, 72), and TSHr (1, 110), have been identified
among causes of congenital hypothyroidism. Most recently, NIS mutations have also been demonstrated to
cause this disease. In the absence of a functional NIS
molecule, I⫺ has no access to the thyroid epithelial cells,
resulting in decreased thyroid hormone biosynthesis (see
sect. I) 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.
Ever since the first case of congenital hypothyroidism due to an I⫺ transport defect was described by Federmann et al. (36), several explanations have been proposed to better define the nature of the defect. However,
the molecular basis of this condition has only begun to be
examined after the cloning of NIS cDNA (21, 105) and the
elucidation of the exon-intron organization of the NIS
gene (106) (Fig. 7). About 48 cases of ITD, belonging to 33
families, have been reported worldwide to date. Seventeen cases from 13 families have been studied at the
molecular level and shown to have a mutation in NIS (see
Table 1).
Shortly after isolation of the cDNA that encodes NIS,
two groups reported almost simultaneously a homozygous missense mutation in patients that had previously
been diagnosed with congenital hypothyroidism caused
by ITD (39, 70) (Table 1, cases 1 and 6, respectively). Both
groups found a DNA substitution of adenine with cytosine
1098
DE LA VIEJA, DOHAN, LEVY, AND CARRASCO
Volume 80
FIG. 7. Correlation of the structural organization of the human NIS (hNIS) gene with the NIS protein. Exons in hNIS
gene are represented with hatched boxes; the 5⬘- and 3⬘-untranslated regions are represented by open boxes. The
putative 13 transmembrane segments in the protein are represented with cylinders. Exons are connected to the
corresponding amino acid regions of the protein by dotted lines (64, 106). Localization of the I⫺ transport defect
mutations is indicated under the schematic representation of the hNIS protein. [Modified from Smanik et al. (106) with
data from Levy et al. (64).]
in nucleotide 1060 in exon 9, resulting in a Pro instead of
Thr at position 354 (T354P) in NIS. The T354P mutation is
located in the putative transmembrane segment IX of the
NIS protein (Figs. 2B, 3, and 7).
The patient in the report of Fujiwara et al. (39) was
homozygous for the T354P mutation and had consanguineous parents; her two siblings were heterozygous for
T354P and were unaffected, suggesting the recessive nature of the disease. Extremely high doses of potassium
iodide treatment maintained this patient euthyroid. HEK293 cells (human embryonic kidney cells) transfected
with the mutant T354P NIS cDNA did not exhibit any I⫺
uptake activity. Based on these results, the authors proposed that T354P probably disrupts (due to the presence
of proline), through a structural change, the putative
transmembrane segment IX of NIS, where the mutation is
located.
The second case (70) was diagnosed 23 years ago
with an I⫺ transport defect (44) (Table 1, case 6). The
patient was euthyroid apparently on account of a very
high iodide dietary intake. However, diffuse goiter with
slightly elevated TSH was noted at 18 yr of age. The
patient exhibited greatly increased NIS mRNA levels
(⬎100-fold) in his thyroid. Hence, these investigators proposed that the observed increase in NIS transcription may
reflect a compensating mechanism for low NIS activity,
possibly related to transcriptional regulation of NIS by I⫺
itself. This patient was born from a consanguineous marriage, and his daughter was heterozygous for the mutation
but had no abnormal phenotype, as would be expected in
a recessive condition.
Subsequently, Levy et al. (65) studied the T354P mutation by site-directed mutagenesis and transfection of
COS cells. They determined that the T354P NIS was not
active, but they observed by immunofluorescence analysis that it was properly targeted to the plasma membrane.
Then various other amino acids substitutions (Ala, Pro,
Cys, Tyr, Ser) at position 354 were explored to determine
the structural change in putative transmembrane segment
IX, leading to the conclusion that the hydroxyl group at
the ␤-carbon of the amino acid residue at position 354 is
essential for NIS function (see sect. II).
Fujiwara et al. (40) have reported four new cases of
patients with the T354P mutation from two families (Table 1, cases 2–5). Case 2 was diagnosed at age 5, treated
with thyroid powder, and thus maintained euthyroid;
however, this patient’s thyroid gland enlarged gradually
and developed multinodular goiter at the age of 14. The
patient’s parents were heterozygous for the T354P mutation without thyroid disorders. The other three patients
reported by Fujiwara et al. (40) are siblings (Table 1, cases
3–5). These patients and their mother were heterozygous
for T354P, but their father was negative for this mutation.
The patients presented a small goiter that developed late
in life with only slight symptoms of hypothyroidism. Even
though the patients were heterozygous for the T354 mutation, they were previously diagnosed as having ITD but
were not screened for other NIS mutations.
Kosugi et al. (55) have reported more cases of T354P
mutations, including three new and four reevaluated
cases with known ITD from five families (Table 1, cases
6 –12). All these cases were homozygous for the T354P
F
M
M
F
F
M
F
F
M
M
M
F
M
F
F
M
F
1986
1958
1968
1966
1963
1937
1942
1975
1978
1971
1989
1974
1947
1984
1985
1958
1958
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
USA
Japan
Japan
Brazil
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Country
Neonatal
3 yr
2 yr
3 mo
3 mo
5 yr
3 yr
10 yr
13 yr
Euthyroid
Euthyroid
6 yr
1 yr
8 mo
1.5 yr
4 mo
22 yr
Hypothyroidism
12 yr (FA)
10 yr
9 yr
3 mo
8 yr (FA)
13 yr (FA)
8 yr
10 yr
13 yr
18 yr
20 yr
6 yr
None
21 yr
1.5 yr
None
16 yr
Goiter
Normal
Retarded
Retarded
Normal
Normal
Retarded
Retarded
Retarded
Normal
Normal
Normal
Retarded
Retarded
Retarded
Retarded
Retarded
Normal
Development
54
54
15, 85
86
⫺
⫺
⫺
⫺
71
91, 126
55
55
55, 70
40
39,
40
40
40
40
44,
55
46,
46,
55
55
55,
54,
Reference
No.
⫹
⫹
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫺
Cons
67
0.8
0.31
0.41
1.42
0.46
1.06
1.4
2.35
0.61
1.06
1.25
0.46
0.39
27
36
12
69
54
91
⬍10
11
1.2
5
10
9
4
37
0.40
T3, ␮g/l
(0.94–1.54)
2
T4, ␮g/l
(50–110)
142
1,044
484
104
730
25
71
110
300
3.3
1.3
13.3
148
37.5
⬎100
430
217
TSH,
mU/l
(0.3–4.0)
3.0
4.0
3.6
1.0
4.5
1.0
1.2
4.8
1.2
2.5
2.0
2.1
1.3
3.0
1.4
2.4
0.9
TRIU, %
(10–40)
3
1.0
1.0
3.0
1.61
0.94
1.1
0.93
1.2
1.5
1.1
1.0
1.0
1.5
2.6
1.8
0.7
S/P ratio
(⬎20)
L-T4
L-T4
12 mg I/day
L-T4
L-T4
12.6 mg I/day
12.6 mg I/day
1 mg I/day
L-T4
L-T4
189 mg I/day
L-T4
A1060C
A1060C
A1060C/?
A1060C/?
A1060C/?
A1060C
A1060C
A1060C
A1060C
A1060C
A1060C
A1060C
G277C
A1060C
G1628A
G1628A
C1163A
C1146G
6
DNA
Mutation
9
9
9
9
9
9
9
9
9
9
9
9
1
9
13
13
6
Exon
13
C1940G
and/or Alt. Spliced
L-T4
Thyroid powder
Thyroid powder
Thyroid powder
Treatment
T354P
T354P
T354P/?
T354P/?
T354P/?
T354P
T354P
T354P
T354P
T354P
T354P
T354P
G93R
T354P
G543E
G543E
C272X
Protein
Mutation
Heterozygous Y531X
515X
Heterozygous Q267E
Homozygous
Homozygous
Heterozygous
Heterozygous
Heterozygous
Homozygous
Homozygous
Homozygous
Homozygous
Homozygous
Homozygous
Homozygous
Heterozygous
Heterozygous
Homozygous
Homozygous
Homozygous
Zygosity
TM IX
TM IX
TM IX
TM IX
TM IX
TM IX
TM IX
TM IX
TM IX
TM IX
TM IX
TM IX
TM III
TM IX
TM XIII
TM XIII
Loop ÷ TM
VII–VIII
Loop ÷ TM
VII–VIII
TM XIII
Loop ÷ TM
XII–XIII
Localization
S/P, salivary to plasma ratio of I; FA, follicular adenoma; empty box, test not done or unknown; Cons, parental consanguinity; TRIU, thyroid radioiodide uptake (%); T4, thyroxine; T3, triiodothyronine; TSH,
thyrotropin; TM, transmembrane segment. Patients 3–5 only have been screened for the T354P mutation.
Sex
Case
Age of Detection
1. Summary of clinical and laboratory parameters of ITD patients and correlation to the NIS molecular defect
Year
of
Birth
TABLE
July 2000
MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
1099
1100
DE LA VIEJA, DOHAN, LEVY, AND CARRASCO
mutation. Whenever the patients’ parents were analyzed,
they were heterozygous for the T354P mutation and had
no obvious thyroid disorders. The first case (case 6) was
previously described by the same group (see above) (70).
Cases 6 and 7, siblings, were euthyroid due to an extremely high I⫺ diet. They presented large diffuse goiters
at ages 18 and 20, respectively. In cases 9 and 12, hypothyroidism was diagnosed early so that L-T4 treatment was
started and goiter development was prevented. The rest
of the cases had different degrees of diffuse goiter and
hypothyroidism. NIS protein determinations were carried
out in cases 6 and 10 and were found to be increased
⬃10-fold with respect to normal tissue. Immunohistochemical staining of NIS in these two patients indicated
that NIS was overexpressed and properly localized in the
basolateral plasma membrane of the thyroid cells (55).
The same group has subsequently reported three patients with ITD from two families, presenting new mutations of the NIS gene (54) (Table 1, cases 13–15). Case 13
had already been reported (91) and reviewed earlier by
Wolff (126). This patient carried compound heterozygous
mutations. From the paternal allele he presented a DNA
substitution of guanidine with cytosine in the 277 nucleotide in exon 1. From the maternal allele he presented a
substitution of adenine with cytosine in nucleotide 1060
in exon 9. These resulted in the compound G93R/T354P
mutation, affecting the putative transmembrane segments
III and IX of NIS (Fig. 7). The patient’s father was heterozygous for the G93R mutation and his mother for the
T354P mutation, both without thyroid disorders. They
were not consanguineous. The patient had developed diffuse goiter at age 15, and he had I⫺ responsive hypothyroidism.
Cases 14 and 15 are siblings. They carried the homozygous substitution of guanidine for adenine in the
1,628 nucleotide in exon 13. The resulting G543E mutation is located in the putative transmembrane segment
XIII of NIS (Fig. 7). The patients’ parents, who were not
consanguineous, were heterozygous for G543E, without
thyroid disorders. The patients’ younger sister did not
carry the mutation. Neither G93R, T354P, G93R/T354P,
nor G543E mutant cDNA transiently transfected into
COS-7 displayed any I⫺ transport activity (54). Nevertheless, these mutations were not characterized at the protein level so that the possibility that these mutations
resulted in a nonfunctional protein or one not properly
targeted to the plasma membrane remains open.
Only two ITD cases characterized at the molecular
level are not from Japan (85, 86) (Table 1, cases 16 and
17). The first one (Table 1, case 16), from Brazil, was also
reviewed by Camargo et al. (15). The patient presented a
substitution of cytosine 1163 with adenine in exon 6,
resulting in a premature stop codon at position 272
(C272X). The mutant NIS protein is truncated in the hydrophilic segment between the putative transmembrane
Volume 80
segments VII and VIII (Fig. 6), and no I⫺ uptake activity
was detected after transfecting mutant NIS cDNA into
COS-7 cells. The patient was homozygous for the mutation. His parents were not consanguineous; his mother,
son, and a paternal aunt were heterozygous for C272X,
and his father was not investigated. All of the carriers
were euthyroid with normal or slightly enlarged thyroid
glands. The patient’s goiter was noted at 3 mo of age and
treated with L-T4, but examination after several years
revealed low T4 and high TSH levels. A nodule developed
in each thyroid lobe. Subsequent treatment with high I⫺
restored the patient to the euthyroid state.
Case 17 in Table 1 was reported by Pohlenz et al.
(86). This was a patient of Hispanic ancestry who carried
a compound heterozygous mutation. From the paternal
allele she presented a substitution of cytosine for guanidine in nucleotide 1146 in exon 6 that resulted in a Gln for
Glu amino acid substitution (Q267E) in NIS. This mutation is located between the putative transmembrane segments VII and VIII of NIS (Fig. 7). COS cells transfected
with the mutated cDNA did not exhibit I⫺ transport activity. The patient’s brother, father, and a paternal uncle
and aunt were heterozygous for the mutation, and they
were clinically unaffected. From the maternal allele she
presented a substitution of cytosine with guanidine in
nucleotide 1940 in exon 13. This point mutation has a
double effect. First, it creates a stop codon at amino acid
531 (Y531X) with the consequent deletion of the entire
hydrophilic COOH terminus (Fig. 7). Second, it generates
a new 3⬘-acceptor splice site for intron 12 that produces a
67-nucleotide deletion in exon 13, rendering a protein
with only 515 amino acids that has a deleted part of the
putative transmembrane segment XIII and of the COOH
terminus of NIS. The in vitro activity of these truncated
proteins was not evaluated. None of the heterozygous
maternal members was affected. This patient was diagnosed with hypothyroidism during neonatal screening
and treated with L-T4. At 12 yr of age she was diagnosed as
having ITD. She presented a nodule that had not been
observed 1 yr before. Histological investigation revealed a
follicular adenoma.
In vitro experiments in cells transfected with the
various NIS mutants found in ITD patients have shown
that the mutant NIS proteins do not elicit I⫺ transport.
However, the precise structural change brought about by
a NIS mutation has only been determined for T354P.
Therefore, it remains to be determined whether the other
NIS mutations give rise to proteins that are nonfunctional
but properly targeted to the plasma membrane, proteins
that are rapidly degraded, or retained in intracellular organelles. It is unknown how I⫺ enters the thyroid follicular cells in these patients. However, a high I⫺ diet has
been shown to be able to maintain some of these patients
in a euthyroid state. It is possible that thyroidal I⫺ transport in these patients occurs by “simple diffusion” or by
July 2000
MOLECULAR ANALYSIS OF THE SODIUM/IODIDE SYMPORTER
nonspecific channels, as suggested by Wolff (126). Moreover, Cl⫺ channels have been shown to be able to transport other halides with different affinities, and in some
cases, these channels display a considerable affinity for I⫺
(26, 27, 52). Administration of high doses of potassium
iodide (KI) (1–200 mg/day) has been reported to maintain
these patients euthyroid, without apparent long-term side
effects.
The inheritance pattern of ITD is autosomal recessive
in all cases. In the investigated families, the heterozygous
carriers were always clinically unaffected, and only a few
of them presented slight goiter. Nevertheless, cases 3–5 in
Table 1, reported by Fujiwara et al. (40), were heterozygous for T354P. However, other mutations could have
been present in these patients and missed in the study
because only T354P was screened.
Most of the patients suffering from this condition
have been from Japan. The high incidence of ITD in the
Japanese population could be related to the high I⫺ diet
prevalent in Japan, which provides an adequate I⫺ supply
even for ITD patients, and allows them to maintain acceptable thyroid hormone synthesis. In a few ITD cases,
expression and activity of the other TSH-regulated thyroid-specific proteins (TPO and Tg) were also investigated. Low or normal levels of Tg protein were observed
in the cases that were studied [Table 1, cases 13 and 16;
and 5 more cases reported by Wolff (126)]. In case 16, Tg
was poorly iodinated, but the patient was euthyroid as a
result of a high I⫺ diet. This indicates that under certain
circumstances “non-NIS mediated transport” is able to
supply enough I⫺ into the follicle to ensure hormonogenesis. Furthermore, Lamas et al. (58) have shown that the
efficiency of hormonogenesis is determined by the available amount of I⫺. Lamas et al. (57) also reported that
under conditions of low I⫺ intake the synthesis of thyroid
hormones on the Tg molecule occurred at the main hormonogenic site, and Passler et al. (83) showed that this
utilization was regulated by TSH.
In two of the patients reported by Wolff (126), TPO
activity was normal or slightly increased. In case 16 (15),
the activity and immunostaining of TPO were threefold
with respect to normal thyroid tissue, probably due to the
high TSH levels. These findings suggest that only NIS is
affected in ITD patients, whereas Tg and TPO expression
and activity remain intact despite the upregulated TSH
levels. Anti-Tg, anti-TPO, and anti-TSHr autoantibodies
were negative when tested in all these patients.
Thyroid morphological findings are fairly heterogeneous, even in patients carrying the same NIS mutation.
Among the patients in which NIS mutations have been
characterized at the molecular levels (as summarized in
Table 1), we find 2 cases without goiter (cases 9 and 12),
11 cases with small or large diffuse goiter (cases 3– 8, 10,
11, and 13–15), 1 case with nodular goiter (case 16), and
3 cases with follicular adenoma (cases 1, 2, and 17). Two
1101
cases (cases 6 and 7) remained euthyroid. Most probably,
this variability reflects the roles played by multiple factors
in the onset and evolution of ITD due to NIS mutations,
including the type of mutation, time of detection of the
condition, time and type of administered therapy, and I⫺
content in the diet. In any case, the considerable strides
made in this area of research in recent years illustrate the
powerful impact that detailed function/structure studies
of this key molecule can have on health and disease.
IX. CONCLUDING REMARKS
The landscape of NIS research has been dramatically
transformed in a very short time. Just six years ago, in
1993, the following was written in a review on “Iodide
Transport in the Thyroid Gland” (16): “The chemical nature of the putative I⫺ carrier has been the subject of
some debate in the past. At one time, phospholipids were
considered potential I⫺ carriers as much as proteins, but
it has long been clear that transport of ions and small
molecules (such as carbohydrates and amino acids)
across cell membranes is mediated by intrinsic membrane
proteins, not phospholipids.” Later in the same article, it
was stated that “the Na⫹/I⫺ symporter molecule has not
been fully characterized yet, as its size, number of subunits, and amino acid sequence remain unknown,” and
with respect to NIS the article concludes by saying that
“there is no question that our understanding of I⫺ translocation processes, both in the thyroid and in other tissues, will be greatly enhanced when this molecule is fully
identified and characterized. Once this is achieved, insight
may subsequently be gained into various important related research fields, given the relevance of I⫺ transport
to Na⫹-dependent symport mechanisms, thyroid physiology and pathophysiology, hormone action mechanisms,
and cell differentiation.” The present review, in fact, focuses on reports that started to appear shortly after publication of the quoted article, covering the isolation of a
cDNA encoding rat NIS, the preparation of anti-NIS antibodies to study NIS topology and its secondary structure,
the examination of the biogenesis and posttranslational
modifications of NIS, its electrophysiological analysis, the
isolation of the cDNA encoding hNIS, the elucidation of
the genomic organization of hNIS, the analysis of NIS
regulation by TSH and I⫺, and the regulation of NIS
transcription, among several other topics. It also discusses spontaneous NIS mutations that have been identified as causes of congenital ITD resulting in hypothyroidism, the roles of NIS in thyroid cancer and thyroid
autoimmune disease, and the expression and regulation
of NIS in extrathyroidal tissues. Perhaps most significantly, the above accomplishments have made it possible
to carry out gene therapy experiments in which the rat
NIS gene has been transduced into various types of hu-
1102
DE LA VIEJA, DOHAN, LEVY, AND CARRASCO
man cells, which then exhibited active iodide transport
and became susceptible to destruction with radioiodide.
Hence, the impact of the continued molecular analysis of
NIS has already reached farther than predicted a few
years ago, given that it extends not only to structure/
function of transport proteins, thyroid pathophysiology,
hormone action mechanisms, and cell differentiation, but
also, quite possibly, to the diagnosis and treatment of
cancer, both in the thyroid and beyond.
We are grateful to Drs. U. Tazebay, C. Riedel, and C. Ginter
for critical reading of the manuscript.
A. De La Vieja was supported in part by Ministry of Education and Science of Spain Grant PF 97 52094152. This project
was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-41544 (to N. Carrasco).
Address for reprint requests and other correspondence: N.
Carrasco, Dept. of Molecular Pharmacology, Albert Einstein
College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461
(E-mail: carrasco@aecom.yu.edu).
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