Post-transcriptional Regulation of the Sodium/Iodide Symporter by Thyrotropin*

Claudia RiedelDagger §, Orlie Levy, and Nancy CarrascoDagger ||

From the Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Na+/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

The Na+/I- 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).

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- 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.

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- 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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- 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.

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- 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 gamma -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.

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- 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 gamma -counter. Data were standardized per mg of protein.

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 -70 °C.

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 -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.

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(-) cells, respectively. Plates were visualized by autoradiography after 3 days at -70 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TSH Differentially Regulates NIS Expression and I- 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.

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- 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.

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%.


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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).

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.


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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.

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).


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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.

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(-)) as calculated by the migration coefficient. These results indicate that NIS is a phosphoprotein and that the NIS phosphorylation pattern is modulated by TSH.


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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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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- 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.

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(-) 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.

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- 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.

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), gamma -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.

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- 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.

The results presented here are highly relevant to thyroid cancer. It is of major diagnostic importance that most thyroid cancers exhibit decreased I- 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.

    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.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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