From the Department of Molecular Pharmacology, Albert
Einstein College of Medicine, Bronx, New York 10461
Received for publication, January 22, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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The Na+/I The Na+/I The iodine-containing thyroid hormones triiodothyronine and thyroxine
play essential roles in promoting the development and maturation of the
nervous system, skeletal muscle, and lungs and in regulating
intermediary metabolism in virtually all tissues. Thyroid-stimulating
hormone (TSH) is the primary hormonal regulator of thyroid function
overall and has long been known to stimulate I Here we provide evidence for post-transcriptional regulation of NIS
function by TSH. Our results show for the first time that NIS is a
phosphoprotein and that the NIS phosphorylation pattern is regulated by
TSH. Furthermore, our data indicate that in the absence of TSH, NIS is
redistributed from the plasma membrane to intracellular compartments.
This suggests that under TSH deprivation, the loss of I Cell Culture--
FRTL-5 rat thyroid cells, kindly provided by
Dr. L. D. Kohn (National Institutes of Health, Bethesda, MD), were
grown in Ham's F-12 media (Life Technologies, Inc.) supplemented with
5% calf serum, 1 mM non-essential amino acids (Life
Technologies, Inc.), 10 mM glutamine, 100 units/ml
penicillin, 100 µg/ml streptomycin, and a six-hormone mixture (6H)
containing insulin (1.3 µM), hydrocortisone (1 µM), transferrin (60 pM),
L-glycyl-histidyl-lysine (2.5 µM), somatostatin (6.1 nM), and TSH (1 milliunits/ml) as
reported previously (23). Cells were grown in a humidified atmosphere
with 5% CO2 at 37 °C. To study the effect of TSH
deprivation, FRTL-5 cells were kept in the same medium without TSH
(5H). FRTL-5 cells are viable in this medium for at least 15 days (23).
TSH was obtained from the National Hormone Pituitary Program, and all
other reagents were purchased from Sigma.
Preparation of Membrane Vesicles (MV)--
MV for
I
MV for immunoblot analysis were prepared as described above except that
the final pellet was resuspended in 250 mM sucrose, 1 mM EGTA, 10 mM Hepes-KOH (pH 7.5).
I
FRTL-5 MV were assayed as described (19). MV were thawed at 37 °C
and placed on ice. Aliquots containing 50 µg of protein (10 µl)
were assayed for 125I Immunoblot Analysis--
SDS-9% polyacrylamide gel
electrophoresis and electroblotting to nitrocellulose were performed as
described previously (12). Samples were diluted 1:2 with loading buffer
and heated at 37 °C for 30 min prior to electrophoresis. Immunoblot
analyses were also carried out as described (12) with 930 pM of affinity-purified anti-NIS polyclonal antibody (Ab)
and 1:1500 of a horseradish peroxidase-linked goat anti-rabbit IgG
(Amersham Pharmacia Biotech). Proteins were visualized by an enhanced
chemiluminescence Western blot detection system (Amersham Pharmacia
Biotech).
Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling and immunoprecipitation were performed as described previously
(12). Briefly, FRTL-5 cells in 60-mm plates kept in the presence or
absence of TSH were washed and incubated for 30 min with cysteine- and
methionine-free RPMI 1640 medium supplemented with dialyzed 5% calf
serum. Cells were labeled with 480 µCi/ml
[35S]methionine/cysteine (Promix, DuPont) for the
indicated times, followed by washes and incubation with regular media
supplemented with 10× methionine/cysteine for the indicated times.
Cells were lysed with 1% SDS in PBS containing aprotinin (90 µg/ml),
leupeptin (4 µg/ml), and PMSF (0.8 mM), followed by a
16-fold dilution with 1% Triton X-100, 1% deoxycholate, 200 mM NaCl, 1% BSA, 50 mM Tris-HCl (pH 7.5).
Preimmune serum and protein G fast flow Sepharose beads (Amersham
Pharmacia Biotech) were added and incubated at 4 °C for 60 min.
Lysate was centrifuged at 100,000 × g for 30 min. Supernatants were incubated with 1:40 dilution of anti-NIS antisera for
60 min at 4 °C, followed by the addition 30% of a slurry of protein
G fast flow Sepharose beads incubated at 4 °C for 60 min. Beads were
centrifuged at 14,000 × g for 5 min and alternately washed with low ionic strength buffer (150 mM NaCl, 1%
Triton X-100, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 7.5)), with high ionic strength buffer (150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EDTA, 0.5 M LiCl, 10 mM Tris-HCl
(pH 7.5)), and with 10 mM Tris-HCl (pH 7.5). Samples were
heated at 37 °C for 30 min in loading buffer prior to SDS
electrophoresis. Gels were fixed and soaked in Fluoro-Hance (Research
Products International). Gels were vacuum-dried and exposed for
autoradiography at Cell Surface Biotinylation--
Cell surface biotinylation was
performed in FRTL-5 cells kept in 6H or 5H medium as a modification of
a method described previously (24). Cells were grown in 12-well plates
to 80% confluence. Cells were washed with PBS/CM (PBS with 0.1 mM CaCl2 and 1 mM MgCl2) and incubated twice for 20 min at 4 °C with 1.5 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-biotin) (Pierce) in 20 mM Hepes (pH 8.5), 2 mM CaCl2, and 150 mM NaCl. Cells
were washed twice for 20 min with PBS/CM-containing 100 mM
glycine at 4 °C and lysed with 1% SDS in 150 mM NaCl, 5 mM EDTA, 1% of Triton X-100, 50 mM Tris (pH
7.5) containing aprotinin (90 µg/ml), leupeptin (4 µg/ml), and PMSF
(0.8 mM), referred to as buffer A. Samples were diluted
10× with buffer A without SDS. Streptavidin-agarose beads (Pierce)
were added to the lysate and incubated overnight at 4 °C. The next
day the lysate was centrifuged at 14,000 × g for 5 min
to separate beads from the supernatant. Beads were washed 3× with
buffer A without SDS and 2× with high ionic strength buffer (500 mM NaCl, 0.1% Triton X-100, 5 mM EDTA, and 50 mM Tris-HCl (pH 7.5)). The final wash was done with 50 mM Tris-HCl (pH 7.5). Beads were resuspended in sample
buffer and heated for 5 min at 75 °C.
Immunofluorescence--
FRTL-5 cells in the presence of TSH were
seeded onto poly-(lysine)-coated coverslips. Forty eight hours after
seeding, cells were changed to 6H or 5H for the indicated times. Cells
were washed 3× with PBS/CM, fixed with 2% paraformaldehyde in
PBS for 20 min at RT, and rinsed with PBS/CM. Cells were permeabilized
with 0.1% Triton in PBS/CM plus 0.2% BSA (PBS/CM/TB) for 10 min at
RT. Cells were quenched with 50 mM NH4Cl in
PBS/CM for 10 min at RT and rinsed with PBS/CM/TB. Cells were incubated
with 8 nM anti-NIS Ab in PBS/CM/TB for 1 h at RT,
washed, and incubated with 1:700 dilution of fluorescein-labeled goat
anti-rabbit Ab (Vector Laboratories). After washing, cells on
the coverslips were mounted onto microscope slides using an antifade
kit from Molecular Probes. Coverslips were sealed with quick-dry nail
polish and allowed to dry in the dark for 2 h at RT and stored at
4 °C. NIS immunofluorescence was analyzed with a Bio-Rad Radiance
2000 Laser Scanning Confocal MRC 600, equipped with a Nikon Eclipse
epifluorescent microscope.
32P in Vivo Labeling--
32P in
vivo labeling was performed as described previously (25). Cells
were grown to 70-80% confluency in 100-mm tissue culture plates and
incubated for 30 min in 4 ml of phosphate-free Dulbecco's modified
Eagle's medium (Sigma) supplemented with 5% calf serum. Then 100 µCi/ml ortho[32P]phosphoric acid (Pi)
(DuPont) was added to the culture medium and incubated for 5 h at
37 °C. Cells were lysed with 1% SDS in PBS containing phosphatase
inhibitors (50 nM calyculin (Sigma), 10 mM NaF,
2 mM EDTA, 4 µM cantharidin (Sigma), 2 mM vanadate, and 100 µM phenylarsyn oxide
(Calbiochem)) and protease inhibitors (3 µg/ml leupeptin, 2 µg/ml
aprotinin, and 0.8 mM PMSF). After lysis, NIS was
immunoprecipitated, subjected to electrophoresis, and
electrotransferred to nitrocellulose. NIS was visualized by autoradiography after 3 h at Two-dimensional Tryptic Phosphopeptide Mapping of in Vivo Labeled
NIS--
The phosphopeptide map was performed as described previously
(25). Tryptic phosphopeptides were separated in two dimensions on
cellulose thin layer plates by electrophoresis at pH 1.9 for 50 min at
650 V, followed by chromatography (1-butanol/pyridine/acetic acid/H2O, 50:33:1:40, v/v). Approximately 1000 and 500 cpm
were loaded onto each plate from TSH(+) and TSH( TSH Differentially Regulates NIS Expression and I TSH Is Required for de Novo NIS Biosynthesis--
To test whether
NIS is synthesized in the absence of TSH, cells that had been deprived
of TSH for 5 days were metabolically labeled for 10 min with
[35S]methionine/cysteine and chased for 8 h. NIS
immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis
showed that de novo biosynthesis of NIS occurred only when
cells were maintained in the presence of TSH (Fig.
2). NIS remained detectable for up to 10 days following TSH deprivation (Fig. 1B), i.e. in
the absence of de novo NIS biosynthesis (Fig. 2),
demonstrating that I NIS Half-life Is Modulated by TSH--
The observation that NIS
remains detectable after prolonged TSH deprivation in the absence of
de novo NIS biosynthesis suggests that NIS has a long
half-life. To determine the precise half-life of NIS and whether it is
modulated by TSH, cells maintained in the presence of TSH were
pulse-labeled with [35S]methionine/cysteine for 5 min and
chased for different times in the presence (Fig.
3A) or absence of TSH (Fig.
3B). As indicated above, NIS migrates as an ~85-kDa broad
band. The ~70-kDa band corresponds to a nonspecific unrelated
polypeptide that, unlike NIS, was also immunoprecipitated by preimmune
serum (not shown). The half-life of NIS was determined to be ~5 days
in the presence and ~3 days in the absence of TSH (Fig.
3C). This indicates that TSH modulates the long half-life of
NIS, increasing it by 40%.
TSH Regulates the Subcellular Distribution of NIS--
To assess
the effect of TSH on NIS content at the plasma membrane, we performed
cell surface biotinylation experiments in the presence of TSH and then
over the course of 10 days after TSH was removed from the culture
medium. To ensure that only polypeptides facing the extracellular
milieu would be biotinylated, we utilized the NH2-specific
and plasma membrane-impermeable biotinylating reagent
Sulfo-NHS-SS-biotin. The entire biotinylated fraction was isolated with
streptavidin-coated beads and was immunoblotted with anti-NIS Ab,
whereas only 1:50 of the non-biotinylated fraction was loaded onto the
gel (Fig. 4, A and
B, respectively). Densitometric quantitation of the bands
showed that NIS content at the plasma membrane decreased over time
after TSH withdrawal in a fashion that correlated very closely with the
corresponding decrease in NIS activity in intact cells (Fig.
4C). While 1 day of TSH deprivation causes a similar
decrease in both intracellular and cell surface NIS, by 3 days after
TSH withdrawal a more pronounced decrease in NIS content was detected
at the plasma membrane than in intracellular compartments (Fig.
4C). This indicates that TSH regulates the subcellular
distribution of NIS.
The possible regulatory role played by TSH in the subcellular
distribution of NIS was further investigated by confocal
immunofluorescence analysis of NIS subcellular localization in response
to TSH withdrawal over a 10-day period (Fig.
5). As anti-NIS Ab recognizes a
cytosol-facing epitope of NIS (i.e. the carboxyl terminus),
cells were fixed and permeabilized prior to incubation with the Ab.
Given that NIS expression decreases after TSH deprivation, images were
taken with different exposure times. In the presence of TSH, FRTL-5 cells predominantly displayed a stark immunofluorescent staining delineating the periphery of the cells, strongly indicative of plasma
membrane localization for NIS (Fig. 5, 0 days). Some
intracellular staining was also observed. The cell surface staining
pattern decreased slowly after removal of TSH, disappearing completely by day 3, at which point only an intracellular pattern remained. The
intracellular NIS pattern observed on day 3 was punctate and spread
throughout the cytoplasm. In contrast, by days 5-10 the NIS punctate
distribution decreased noticeably and was localized further from the
perinuclear region. Immunofluorescence was abolished by preincubation
of the Ab with antigen peptide or when the second but not the first Ab
was added, indicating that the observed staining is specific for NIS
(12). These observations are consistent with the biotinylation findings
described above (Fig. 4), suggesting that TSH is required for NIS
localization at the cell surface. Therefore, the absence of TSH over
time causes NIS to mainly redistribute to and/or remain in
intracellular compartments. These data support the notion that in
addition to regulating NIS expression, TSH also regulates the
subcellular distribution of NIS. In the absence of TSH, not only is
de novo NIS biosynthesis nonexistent (Fig. 2) but NIS is
increasingly re-distributed from the plasma membrane to intracellular
compartments over time (within the 3-7-day range).
TSH Modulates NIS Phosphorylation--
The mechanism by which
TSH regulates the subcellular distribution of NIS is unknown.
Phosphorylation has been shown to be implicated in activation and
subcellular distribution of several transporters (27-32). NIS has
several consensus sites for kinases, including those for
cAMP-dependent protein kinase, protein kinase C, and CK-2.
Furthermore, TSH actions in the thyroid are mainly mediated by cAMP,
raising the possibility that phosphorylation might be involved in the
regulation of NIS distribution. FRTL-5 cells were labeled with
32Pi for 5 h and lysed. NIS was
immunoprecipitated with anti-NIS Ab, and the immunoprecipitate was
subjected to electrophoresis. The autoradiogram revealed that NIS was
phosphorylated, independently of the presence of TSH in the culture
medium (Fig. 6A). Given the
decreased expression of NIS in TSH-deprived cells, the amount of
32P-NIS was considerably lower in these cells than in those
grown in the presence of the hormone. To assess whether NIS
phosphorylation is modulated by TSH, we performed
32Pi labeling in the presence or absence of
TSH, and immunoprecipitated 32P-labeled NIS was subjected
to digestion with trypsin as described under "Experimental
Procedures." The phosphopeptide map obtained when TSH was present was
markedly different from that when TSH was absent (Fig. 6B).
Five phosphopeptides were resolved in the presence and three in the
absence of TSH. Only one among these eight phosphopeptides seemed to be
common to both conditions (number two for TSH(+) and number eight for
TSH( The regulation of membrane transport proteins is a highly complex
process that takes place at various levels (33-35). Here we show that
this is the case for NIS regulation. NIS, being the transporter that
mediates the first step (i.e. active I With our high affinity anti-NIS Ab we demonstrated conclusively by
immunoblot analysis that NIS is present in FRTL-5 cells as late as 10 days after TSH withdrawal (Fig. 1B) and that de novo NIS biosynthesis requires TSH (Fig. 2). Therefore, it is clear that any NIS molecules detected in TSH( Several transporters are modulated by post-transcriptional regulation
of their trafficking to the plasma membrane and/or by internalization
from the plasma membrane to intracellular compartments (37, 38). For
example, the glucose transporter 4 (GLUT4) (35) is targeted to the
plasma membrane in response to insulin, whereas the serotonin
transporter is internalized in the presence of its antagonist cocaine
(27). Therefore, it seems feasible that regulation of the subcellular
distribution of NIS might also be a mechanism involved in modulating
I The precise mechanism by which TSH regulates NIS distribution remains
to be fully explored. NIS exhibits several consensus sites for the
cAMP-dependent protein kinase, protein kinase C, and CK-2
kinases. We have observed that NIS is phosphorylated (Fig.
6A) and that the NIS phosphorylation pattern differs when cells are in the presence as compared to the absence of TSH (Fig. 6B). This demonstrates that TSH modulates NIS
phosphorylation. Therefore, given that phosphorylation has been
reported to play a role in regulating targeting of other transporters,
such as the serotonin (27), vesicular monoamine (29), vesicular
acetylcholine (30), The multifaceted TSH-NIS regulatory interaction shown here represents a
key link in the negative feedback loop involving TSH and the thyroid
hormones. First, the mentioned TSH actions on NIS lead, by different
but mutually reinforcing mechanisms (i.e. transcriptional
and post-transcriptional), to stimulation of I The results presented here are highly relevant to thyroid cancer. It is
of major diagnostic importance that most thyroid cancers exhibit
decreased I
symporter (NIS) is a key plasma membrane glycoprotein that mediates
active I
transport in the thyroid gland (Dai, G., Levy,
O., and Carrasco, N. (1996) Nature 379, 458-460), the
first step in thyroid hormone biogenesis. Whereas relatively little is
known about the mechanisms by which thyrotropin (TSH), the main
hormonal regulator of thyroid function, regulates NIS activity,
post-transcriptional events have been suggested to play a role
(Kaminsky, S. M., Levy, O., Salvador, C., Dai, G., and Carrasco,
N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3789-3793). Here we show that TSH induces de novo NIS
biosynthesis and modulates the long NIS half-life (~5 days). In
addition, we demonstrate that TSH is required for NIS targeting to or
retention in the plasma membrane. We further show that NIS is a
phosphoprotein and that TSH modulates its phosphorylation pattern.
These results provide strong evidence of the major role played by
post-transcriptional events in the regulation of NIS by TSH. Beyond
their inherent interest, it is also of medical significance that these
TSH-dependent regulatory mechanisms may be altered in the
large proportion of thyroid cancers in which NIS is predominantly
expressed in intracellular compartments, instead of being properly
targeted to the plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
symporter
(NIS)1 is an intrinsic plasma
membrane protein that mediates the active transport of I
in the thyroid and other tissues such as salivary glands, gastric mucosa, and lactating mammary gland (1, 2). NIS is of central significance in thyroid pathophysiology as the route by which I
reaches the gland for thyroid hormone biosynthesis and
as a means for diagnostic scintigraphic imaging and for radioiodide
therapy in thyroid cancer (3). NIS couples the inward translocation of
Na+ down its electrochemical gradient to the simultaneous
inward translocation of I
against its electrochemical
gradient (4-6) with a 2:1 Na+/I
stoichiometry (6). Cloning and sequencing of the rat NIS cDNA revealed a protein of 618 amino acids (7), which is highly homologous
(87% identity) to the subsequently cloned human NIS (8). The current
secondary structure model depicts NIS as a protein with 13 transmembrane segments, the amino terminus facing the extracellular
side and the carboxyl terminus facing the cytosol, both of which we
have demonstrated experimentally (9).
uptake
activity in the thyroid (10). No thyroidal I
uptake is
detected in humans whose serum TSH levels are suppressed (11). In
addition, up-regulation of NIS thyroid expression and I
uptake activity by TSH has been demonstrated in rats in vivo (12), in the rat thyroid-derived FRTL-5 cell line (13), and in human thyroid primary cultures (14, 15). TSH up-regulates I
uptake activity by a cAMP-mediated increase in NIS
transcription (13, 16-18). After TSH withdrawal a reduction of both
intracellular cAMP levels and I
uptake activity is
observed in FRTL-5 cells. This is a reversible process, as
I
uptake activity can be restored either by TSH or agents
that increase cAMP (13, 18). I
uptake activity
surprisingly persists in membrane vesicles (MV) prepared from FRTL-5
cells that, when intact, have completely lost I
uptake
activity due to prolonged TSH deprivation (19). This suggests that
mechanisms other than transcriptional might also operate to regulate
NIS activity in response to TSH.
transport activity in FRTL-5 cells is due to NIS intracellular distribution. Interestingly and contrary to expectations, NIS is
overexpressed in some thyroid cancers, notwithstanding their decreased
I
uptake activity (20,
21).2 Moreover, overexpressed
NIS in these cells is predominantly retained intracellularly.2 The intracellular NIS redistribution
pattern that we observed in FRTL-5 cells maintained in the absence of
TSH resembles that reported in thyroid tumors, underscoring the
importance of elucidating the mechanisms that govern the subcellular
localization of NIS.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
transport were prepared as described previously (19).
Briefly, FRTL-5 cells kept in TSH(+) or TSH(
) medium were washed,
harvested, and resuspended in ice-cold 250 mM sucrose, 1 mM EGTA, 10 mM Hepes-KOH (pH 7.5), containing
aprotinin (90 µg/ml) (Roche Molecular Biochemicals), leupeptin (4 µg/ml) (Roche Molecular Biochemicals), and phenylmethanesulfonyl
fluoride (PMSF) (0.8 mM) (Sigma). Cells were disrupted with
a motor-driven Teflon pestle homogenizer. The homogenate was
centrifuged twice at 500 × g for 15 min at 4 °C,
and the supernatant was centrifuged at 100,000 × g for
1 h at 4 °C. The pellet was resuspended in ice-cold 250 mM sucrose, 1 mM MgCl2, 10 mM Hepes-KOH (pH 7.5), aliquoted, and stored in liquid nitrogen.
Transport in Intact Cells and
MV--
I
transport assays in intact cells were
performed with 90% confluent FRTL-5 cells in 12-well plates that were
kept either in 6H or 5H medium (23). Briefly, after aspirating the
culture medium, cells were washed two times with 0.5 ml of modified
Hanks' balanced salt solution (HBSS). Cells were incubated with HBSS buffer containing 20 µM Na125I (specific
activity 50 Ci/mol) for 45 min at 37 °C in a humidified atmosphere
with 5% CO2. Reactions were terminated by aspirating the
radioactive solution and washing three times with cold HBSS. Intracellular 125I
was released by
permeabilizing the cells with 500 µl of 95% cold ethanol and was
quantitated in a
-counter. DNA in each well was determined by the
diphenylamine method (19). I
uptake was expressed as
picomoles of I
per µg of DNA in each well.
uptake by incubating at
room temperature (RT) with an equal volume (10 µl) of a solution
containing 20 µM Na125I (specific activity
1.1 Ci/mmol), 1 mM MgCl2, 10 mM
Hepes-KOH (pH 7.5), 2 mM methimazole, 200 mM
NaCl, 30 µM NaClO4. Reactions were terminated
at the 30-s time point by the addition of 3 ml of ice-cold quenching
solution: 250 mM KCl, 1 mM methimazole, and 1 mM Tris-HCl (pH 7.5), followed by rapid filtration through wet nitrocellulose filters (0.45-µm pore diameter). Radioactivity retained by MV was determined by quantitating filters in
-counter. Data were standardized per mg of protein.
70 °C.
70 °C. The NIS band was excised from the nitrocellulose and digested with trypsin as described (26).
Briefly, nitrocellulose strips were treated with 0.5% polyvinylpyrrolidone-30 in 100 nM acetic acid for 30 min,
washed with water, and digested with
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (10 µg) (Worthington) in 100 mM
NH4HCO3 (pH 8.2), 1 mM
CaCl2 for 24 h at 37 °C. Under these conditions, ~60% of the 32P was released from the nitrocellulose.
) cells,
respectively. Plates were visualized by autoradiography after 3 days at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Uptake Activity in FRTL-5 Cells--
We measured
Na+-dependent, perchlorate-inhibitable
(i.e. NIS-mediated) I
uptake activity in
intact FRTL-5 cells over the course of 10 days after TSH was removed
from the culture medium and in MV prepared from these cells (Fig.
1A). MV are a pool of sealed
vesicles from all subcellular compartments except the nuclear membrane.
As reported previously (19), while I
transport activity
decreased by 75% in intact cells 3 days after removal of TSH (Fig.
1A, empty bars), I
transport activity only
decreased by 25% in MV (Fig. 1A, filled bars). By 5 days
after TSH withdrawal, I
uptake was completely abolished
in intact cells, whereas in MV it was still as high as 60% of the
initial activity. To determine whether the reduction of I
uptake in intact cells was due to a decrease in NIS expression, we
subjected MV from these cells to immunoblot analysis with anti-NIS Ab
(Fig. 1B), and we monitored the ~85-kDa broad band
corresponding to fully glycosylated NIS (12). Although NIS expression
decreased to ~50% of its initial level after 3 days of TSH
deprivation, NIS expression in MV remained detectable after 7-10 days
(Fig. 1B), i.e. even after I
uptake
in intact cells was completely abolished (Fig. 1A). That I
transport activity in MV from TSH-deprived cells
persists during the entire time course is consistent with NIS
expression in these cells.
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Fig. 1.
TSH regulates I transport and
NIS expression in FRTL-5 cells. A, I
transport activity. FRTL-5 cells were kept in the presence or absence
of TSH for the indicated number of days. I
transport was
measured in intact cells (empty bars) and in membrane
vesicles (MV) prepared from these cells (filled
bars). I
transport measured in cells maintained in
the presence of TSH and in their MV was defined as 100%.
I
transport corresponding to days 1, 3, 5, 7, and 10 after TSH removal was expressed as the percentage of I
transport relative to day 0. The values represent the means ± S.E. of at least four independent experiments performed in triplicate.
B, NIS expression in FRTL-5 cells. MV from FRTL-5 cells were
prepared, electrophoresed, and analyzed by Western blot using a high
affinity anti-NIS Ab as described under "Experimental Procedures."
The NIS protein corresponds to the ~85-kDa broad band.
uptake observed in MV from
TSH-deprived cells is mediated by NIS molecules synthesized prior to
TSH removal.
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Fig. 2.
De novo synthesis of NIS requires
TSH. FRTL-5 cells maintained in the presence or absence of TSH for
5 days were metabolically labeled with 480 µCi/ml
[35S]methionine/cysteine for 10 min. Cells were chased
for 8 h and lysed. NIS was immunoprecipitated with anti-NIS Ab and
electrophoresed. The ~85-kDa broad band corresponds to the NIS
monomer and the ~160-kDa to the dimer.
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Fig. 3.
NIS has a long half-life, which is modulated
by TSH. To determine the half-life of NIS, FRTL-5 cells were
pulsed for 5 min with 480 µCi/ml of
[35S]methionine/cysteine in the presence of TSH. During
the chase period an aliquot of cells was maintained in the presence of
TSH (A) and a second aliquot kept in the absence of TSH
(B). Chase periods are indicated in the horizontal
axis. Samples were processed as described in Fig. 2. NIS bands
were subjected to densitometric analysis (NIH program) for quantitation
(TSH( ), circles and continuous line, TSH (+),
squares and dotted line). C,
inset, scatter plot of NIS half-life from three independent
experiments in the presence (+) and absence (
) of TSH. Student's
t test (unpaired) yielded p < 0.0001. Data
fitting, S.D., and Student's t test calculations were done
with the PrismTM 2.0 software (GraphPad, San Diego,
CA).
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Fig. 4.
NIS at the cell surface decreases in close
correlation with I transport after TSH withdrawal.
Cell surface biotinylation experiments were performed in FRTL-5 cells
that were kept in the presence or absence of TSH. Cells were
biotinylated with Sulfo- NHS-SS-biotin, a membrane-impermeable
reagent, and lysed, and biotinylated proteins were separated from
non-biotinylated proteins by precipitation with streptavidin. The
membrane impermeability of Sulfo-NHS-SS-biotin was verified by
demonstrating that the intracellular protein actin was not biotinylated
(not shown). All biotinylated proteins (A) and 1:50 of the
supernatant containing the non-biotinylated intracellular proteins
(B) were electrophoresed, electrotransferred to
nitrocellulose, and immunoblotted with anti-NIS Ab. Equivalent protein
amounts were loaded on each lane, as assessed by immunoblot analysis
with anti-actin Ab (data no shown). Hence, A shows NIS
content at the plasma membrane, and B shows 1:50 of NIS
content in intracellular compartments. C, NIS bands from
both immunoblots were subjected to densitometric analysis (NIH program)
for quantitation (squares, biotinylated NIS;
asterisks, non-biotinylated NIS), and the results were
plotted along with the corresponding I
transport activity
values from intact cells (circles). All values were
expressed as percentage relative to day 0. Values represent the
means ± S.E. of at least three independent experiments.
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Fig. 5.
NIS is redistributed to intracellular
compartments during TSH deprivation. NIS staining was performed in
FRTL-5 cells with anti-NIS Ab. Cells were maintained in the presence or
absence of TSH for the indicated number of days. NIS immunofluorescence
in these cells was analyzed by confocal microscopy as described under
"Experimental Procedures." Magnification was × 60.
)) as calculated by the migration coefficient. These results
indicate that NIS is a phosphoprotein and that the NIS phosphorylation
pattern is modulated by TSH.
View larger version (38K):
[in a new window]
Fig. 6.
NIS is a phosphoprotein, and its
phosphorylation pattern is modulated by TSH. FRTL-5 cells were
grown to 70% confluence in 100-mm tissue culture plates in the
presence or absence of TSH for 5 days. Cells were labeled in
vivo with 100 µCi/ml 32P for 5 h at 37 °C.
Cells were lysed, and NIS was immunoprecipitated with anti-NIS Ab,
electrophoresed, and electrotransferred to nitrocellulose.
32P-labeled NIS was visualized after autoradiography at
70 °C for 3 h (A). NIS bands were excised from the
nitrocellulose and digested with trypsin. Tryptic phosphopeptides were
separated in two dimensions on cellulose thin layer plates by
electrophoresis (pH 1.9) for 50 min at 650 V, followed by thin layer
chromatography. Phosphopeptides were visualized by autoradiography
(B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
uptake)
in thyroid hormone biosynthesis, provides a suitable regulatory target
for TSH, which is the primary hormonal regulatory factor of thyroid
function overall. It has long been clear that TSH stimulates thyroidal
I
uptake by up-regulating NIS transcription via cAMP (13,
17, 18). Our findings provide convincing experimental evidence that TSH
also regulates NIS by post-transcriptional mechanisms.
) FRTL-5 cells had to be
synthesized prior to TSH withdrawal. This is consistent with NIS being
a protein with an exceptionally long half-life, as suggested previously
(17, 36). Indeed, by pulse-chase analysis we determined that NIS
half-life is ~5 days in the presence and ~3 days in the absence of
TSH (Fig. 3). Even though the NIS half-life in the absence of TSH is
40% shorter than in the presence of the hormone, it is still
sufficiently long to account for the persistence of significant
I
uptake activity in MV from cells deprived of TSH (Fig.
1). It was the detection of this vesicular activity that first led to the suggestion that NIS might be regulated post-transcriptionally (19),
a notion further supported by several subsequent reports (17, 36). In
addition, it has recently been shown (14) that TSH markedly stimulates
NIS mRNA and protein levels in both monolayer and follicle-forming
human primary culture thyrocytes, whereas significant stimulation of
I
uptake is observed only in follicles, suggesting that
NIS may be regulated by such post-transcriptional events as subcellular distribution.
uptake. We have shown a remarkably close correlation
between NIS plasma membrane content and NIS activity (Fig.
4C), demonstrating that the progressive loss of NIS activity
after TSH withdrawal is due to a decrease in the amount of
NIS present at the cell surface. Furthermore, we observed that 3 days
after TSH deprivation, intracellular NIS decreases at a slower rate
than plasma membrane NIS (compare Fig. 4, A and
B). These data support the notion that active NIS molecules,
initially located in the plasma membrane while TSH is present, are
redistributed to intracellular compartments in response to TSH
withdrawal despite the lack of de novo NIS synthesis and the
40% reduction of the NIS half-life. This model explains the presence
of NIS activity in MV from cells deprived of TSH that, when intact,
exhibit no NIS activity. Clearly, TSH regulates I
uptake
by modulating the subcellular distribution of NIS, without apparently
influencing the intrinsic functional status of the NIS molecules, as
proposed previously (19). In conclusion, TSH not only stimulates NIS
transcription and biosynthesis, it is also required for targeting NIS
to and/or retaining it at the plasma membrane. Future experiments might
distinguish between these two possibilities.
-aminobutyric acid (28), organic cation (OCT1)
(31), and hepatocyte organic anion transporters (32), it will be of considerable interest to investigate whether NIS phosphorylation plays
a role in NIS targeting as well.
uptake
resulting in higher thyroid hormone production and release. Then, a
rise in thyroid hormone circulating levels ultimately inhibits TSH
release in the pituitary gland, and this decreases I
uptake in the thyroid.
uptake relative to the surrounding tissue on
scintigraphy (11). Conversely, the ability of thyroid cancer cells to
sufficiently transport I
is the basis for radioiodide
therapy to be effective against remnant thyroid malignant cells or
metastasis after thyroidectomy. Because of the decrease in
I
uptake observed in thyroid cancer, it had long been
expected that NIS expression would be decreased in thyroid cancer
cells. However, NIS has surprisingly been shown in numerous thyroid
cancers to be actually overexpressed but retained
intracellularly.2 This suggests that malignant
transformation of thyroid cells interferes with the distribution of NIS
to the plasma membrane. Interestingly, we have also observed both
plasma membrane and intracellular NIS expression in breast cancer (1).
The research presented here provides insight into the
post-transcriptional mechanisms involved in the regulation of NIS by
TSH. These are some of the very mechanisms that may be affected in
thyroid cancer. Whereas several researchers have focused on finding
ways to induce NIS transcription in thyroid cancer (22, 39), our
findings indicate that an understanding of the regulatory processes of NIS biosynthesis, targeting, and trafficking is necessary for the
development of complementary strategies to enhance the I
transport ability of thyroid cancers and increase the effectiveness of
radioiodide therapy in these cases.
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ACKNOWLEDGEMENTS |
---|
We are grateful Drs. A. De la Vieja, O. Dohán, M. Amzel, and A. Kalergis for their valuable contributions, insightful discussions, and critical reading of the manuscript. We thank Drs. J. Glavy and G. Orr for help and advice with phosphorylation experiments. We also thank Drs. M. Charron, P. Arvan, E. B. Katz, C. Harley, and A. Dasgupta for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by the National Institutes of Health Grant DK-41544 (to N. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the United States Army Medical Research and Materiel Command Office Award BC990754.
¶ Supported by the National Institutes of Health Hepatology Research Training Grant DK-07218. Current address: Wyeth-Lederle Vaccines, 401 North Middletown Rd. 180/216-17, Pearl River, NY 10965.
To whom correspondence should be addressed: Dept. of Molecular
Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park
Ave., Bronx, New York 10461. Tel.: 718-430-3523; Fax: 718-430-8922; E-mail: carrasco@aecom.yu.edu.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M100561200
2 Dohán, O., Baloch, Z., Banrevi, Z., Livolsi, V., and Carrasco, N. (2001) JCEM 86, in press.
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ABBREVIATIONS |
---|
The abbreviations used are:
NIS, Na+/I symporter;
TSH, thyroid-stimulating
hormone;
MV, membrane vesicles;
PMSF, phenylmethanesulfonyl fluoride;
HBSS, Hanks' balanced salt solution;
Ab, antibody;
PBS, phosphate-buffered saline;
RT, room temperature;
Sulfo-NHS-SS-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tazebay, U. H., Wapnir, I. L., Levy, O., Dohan, O., Zuckier, L. S., Zhao, Q. H., Deng, H. F., Amenta, P. S., Fineberg, S., Pestell, R. G., and Carrasco, N. (2000) Nat. Med. 6, 871-878[CrossRef][Medline] [Order article via Infotrieve] |
2. |
De la Vieja, A.,
Dohan, O.,
Levy, O.,
and Carrasco, N.
(2000)
Physiol. Rev.
80,
1083-1105 |
3. | Mazaferri, E. L. (2000) in The Thyroid: A Fundamental and Clinical Text (Braverman, L. E. , and Utiger, R. D., eds), 8th Ed , pp. 904-930, J. B. Lippincott, Philadelphia |
4. | Bagchi, N., and Fawcett, D. M. (1973) Biochim. Biophys. Acta 318, 235-251[Medline] [Order article via Infotrieve] |
5. | Weiss, S. J., Philp, N. J., and Grollman, E. F. (1984) Endocrinology 114, 1108-1113[Abstract] |
6. |
Eskandari, S.,
Loo, D. D.,
Dai, G.,
Levy, O.,
Wright, E. M.,
and Carrasco, N.
(1997)
J. Biol. Chem.
272,
27230-27238 |
7. | Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460[CrossRef][Medline] [Order article via Infotrieve] |
8. | Smanik, P. A., Liu, Q., Furminger, T. L., Ryu, K., Xing, S., Mazzaferri, E. L., and Jhiang, S. M. (1996) Biochem. Biophys. Res. Commun. 226, 339-345[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Levy, O.,
De la Vieja, A.,
Ginter, C. S.,
Riedel, C.,
Dai, G.,
and Carrasco, N.
(1998)
J. Biol. Chem.
273,
22657-22663 |
10. | Vassart, G., and Dumont, J. E. (1992) Endocr. Rev. 13, 596-611[Medline] [Order article via Infotrieve] |
11. | Martino, E., Bartalena, L., and Pinchera, A. (2000) in The Thyroid: A Fundamental and Clinical Text (Braverman, L. E. , and Utiger, R. D., eds), 8th Ed , pp. 762-773, J. B. Lippincott, Philadelphia |
12. |
Levy, O.,
Dai, G.,
Riedel, C.,
Ginter, C. S.,
Paul, E. M.,
Lebowitz, A. N.,
and Carrasco, N.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5568-5573 |
13. | Weiss, S. J., Philp, N. J., Ambesi-Impiombato, F. S., and Grollman, E. F. (1984) Endocrinology 114, 1099-1107[Abstract] |
14. |
Kogai, T.,
Curcio, F.,
Hyman, S.,
Cornford, E. M.,
Brent, G. A.,
and Hershman, J. M.
(2000)
J. Endocrinol.
167,
125-135 |
15. |
Saito, T.,
Endo, T.,
Kawaguchi, A.,
Ikeda, M.,
Nakazato, M.,
Kogai, T.,
and Onaya, T.
(1997)
J. Clin. Endocrinol. & Metab.
82,
3331-3336 |
16. | Marcocci, C., Cohen, J. L., and Grollman, E. F. (1984) Endocrinology 115, 2123-2132[Abstract] |
17. |
Kogai, T.,
Endo, T.,
Saito, T.,
Miyazaki, A.,
Kawaguchi, A.,
and Onaya, T.
(1997)
Endocrinology
138,
2227-2232 |
18. |
Ohno, M.,
Zannini, M.,
Levy, O.,
Carrasco, N.,
and Di Lauro, R.
(1999)
Mol. Cell. Biol.
19,
2051-2060 |
19. | Kaminsky, S. M., Levy, O., Salvador, C., Dai, G., and Carrasco, N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3789-3793[Abstract] |
20. | Arturi, F., Russo, D., Giuffrida, D., and Schlumberger, M. (2000) Eur. J. Endocrinol. 143, 623-627[Medline] [Order article via Infotrieve] |
21. |
Saito, T.,
Endo, T.,
Kawaguchi, A.,
Ikeda, M.,
Katoh, R.,
Kawaio, A.,
Muramatsu, A.,
and Onaya, T.
(1997)
J. Clin. Invest.
101,
1296-1300 |
22. | Schmutzler, C., and Kohrle, J. (2000) Thyroid 10, 393-406[Medline] [Order article via Infotrieve] |
23. | Weiss, S. J., Philp, N. J., and Grollman, E. F. (1984) Endocrinology 114, 1090-1098[Abstract] |
24. | Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Orr, G. A.,
Han, E. K.,
Browne, P. C.,
Nieves, E.,
O'Connor, B. M.,
Yang, C. P.,
and Horwitz, S. B.
(1993)
J. Biol. Chem.
268,
25054-25062 |
26. | Luo, K. X., Hurley, T. R., and Sefton, B. M. (1991) Methods Enzymol. 201, 149-152[Medline] [Order article via Infotrieve] |
27. |
Ramamoorthy, S.,
and Blakely, R. D.
(1999)
Science
285,
763-766 |
28. |
Law, R. M.,
Stafford, A.,
and Quick, M. W.
(2000)
J. Biol. Chem.
275,
23986-23991 |
29. |
Krantz, D. E.,
Peter, D.,
Liu, Y.,
and Edwards, R. H.
(1997)
J. Biol. Chem.
272,
6752-6759 |
30. |
Krantz, D. E.,
Waites, C.,
Oorschot, V.,
Liu, Y.,
Wilson, R. I.,
Tan, P. K.,
Klumperman, J.,
and Edwards, R. H.
(2000)
J. Cell Biol.
149,
379-396 |
31. |
Mehrens, T.,
Lelleck, S.,
Cetinkaya, I.,
Knollmann, M.,
Hohage, H.,
Gorboulev, V.,
Boknik, P.,
Koepsell, H.,
and Schlatter, E.
(2000)
J. Am. Soc. Nephrol.
11,
1216-1224 |
32. |
Glavy, J. S.,
Wu, S. M.,
Wang, P. J.,
Orr, G. A.,
and Wolkoff, A. W.
(2000)
J. Biol. Chem.
275,
1479-1484 |
33. |
Bradbury, N. A.
(1999)
Physiol. Rev.
79,
175-191 |
34. |
Kopito, R. R.
(1999)
Physiol. Rev.
79,
167-173 |
35. |
Pessin, J. E.,
Thurmond, D. C.,
Elmendorf, J. S.,
Coker, K. J.,
and Okada, S.
(1999)
J. Biol. Chem.
274,
2593-2596 |
36. |
Paire, A.,
Bernier-Valentin, F.,
Selmi-Ruby, S.,
and Rousset, B.
(1997)
J. Biol. Chem.
272,
18245-18249 |
37. |
Brown, D.
(2000)
Am. J. Physiol.
278,
F192-F201 |
38. | Blakely, R. D., and Bauman, A. L. (2000) Curr. Opin. Neurobiol. 10, 328-336[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Venkataraman, G. M.,
Yatin, M.,
Marcinek, R.,
and Ain, K. B.
(1999)
J. Clin. Endocrinol. & Metab.
84,
2449-2457 |