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. 2011 Nov 22;153(1):512–521. doi: 10.1210/en.2011-1548

TSH Regulates Pendrin Membrane Abundance and Enhances Iodide Efflux in Thyroid Cells

Liuska Pesce 1, Aigerim Bizhanova 1, Juan Carlos Caraballo 1, Whitney Westphal 1, Maria L Butti 1, Alejandro Comellas 1, Peter Kopp 1,✉
PMCID: PMC3249672  PMID: 22109890

Abstract

Thyroid hormones are essential for normal development and metabolism. Their synthesis requires transport of iodide into thyroid follicles. The mechanisms involving the apical efflux of iodide into the follicular lumen are poorly elucidated. The discovery of mutations in the SLC26A4 gene in patients with Pendred syndrome (congenital deafness, goiter, and defective iodide organification) suggested a possible role for the encoded protein, pendrin, as an apical iodide transporter. We determined whether TSH regulates pendrin abundance at the plasma membrane and whether this influences iodide efflux. Results of immunoblot and immunofluorescence experiments reveal that TSH and forskolin rapidly increase pendrin abundance at the plasma membrane through the protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin membrane abundance correlates with a decrease in intracellular iodide as determined by measuring intracellular 125iodide and can be inhibited by specific blocking of pendrin. Elimination of the putative protein kinase A phosphorylation site T717A results in a diminished translocation to the membrane in response to forskolin. These results demonstrate that pendrin translocates to the membrane in response to TSH and suggest that it may have a physiological role in apical iodide transport and thyroid hormone synthesis.

The ability of the thyroid to concentrate iodide is essential for the synthesis of thyroid hormones (1–5). It involves transport of iodide through the basolateral membrane into thyroid follicular cells, transport across the cell, and efflux at the apical membrane into the follicular lumen (2, 4, 6). Basolateral iodide uptake is mediated by the sodium-iodide symporter (NIS) and has been characterized in detail (2, 7–10). However, iodide efflux to the follicular lumen is less well understood (2, 8, 11, 12).

TSH is the main regulatory hormone of thyroid cell growth and function and has long been known to have a biphasic effect on thyroidal iodide transport (1, 4, 12, 13). Initially, TSH rapidly stimulates iodide efflux to the follicular lumen, followed by an increase in iodide uptake at the basolateral membrane via cAMP generation and protein synthesis (1, 4, 8, 11–13). Although it is known that TSH increases iodide uptake by transcriptional and posttranscriptional regulation of NIS (9, 10, 14), the mechanisms resulting in the rapid increase of iodide efflux to the follicular lumen have not been elucidated.

Pendred syndrome (PDS), an autosomal recessive disorder caused by homozygous or compound heterozygous mutations in the SLC26A4 (PDS) gene, is characterized by congenital sensorineural deafness, goiter, and impaired iodide organification (15–17). The SLC26A4 gene encodes pendrin, a member of the solute carrier family 26A, which contains several multifunctional anion transporters (18, 19). Functional studies in Xenopus oocytes demonstrated that pendrin is a chloride and iodide transporter (20). In the thyroid, pendrin is located at the apical membrane of thyroid follicular cells (21–23). Functional studies in heterologous cell systems demonstrate that pendrin is able to mediate iodide efflux (21–23), and we reported that pendrin mediates apical iodide efflux in a polarized cell system (24). These observations suggest that pendrin could be involved in vectorial iodide transport at the apical membrane. However, the physiological role of pendrin as an apical iodide transporter has been questioned because of its distinct role as a chloride/bicarbonate exchanger in tissues such as the kidney and the inner ear (25–27) and the absence of an overt thyroid phenotype in the Slc26a4 knockout mouse (7), even under conditions of iodine deficiency (28, 29).

Although previous reports show that TSH induces rapid increase in iodide efflux in polarized and nonpolarized thyroid cells (11, 12), the underlying mechanism has not been elucidated. In this study, we set out to determine whether TSH regulates the insertion of pendrin into the plasma membrane thereby possibly modulating iodide efflux from thyroid cells.

Materials and Methods

Materials

Bovine TSH, forskolin (FSK), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemicals (St. Louis, MO). H-89 and bisindolylmaleimide I (BIS) were purchased from Calbiochem (La Jolla, CA). The antiprotein kinase A (PKA) substrate (RRXS*/T*) antibody was purchased from Cell Signaling Technology (Danvers, MA). 125I was purchased from PerkinElmer (Boston, MA). Poly-D-Lysine cellware two-well culture slides were from BD Biosciences (Bedford, MA). Horseradish peroxidase (HRP)-labeled goat antichicken IgY and Preciphen (agarose-coated goat antichicken IgY) were purchased from Aves Labs (Tigard, OR). A polyclonal rabbit E-cadherin antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other reagents were commercial products of the highest grade available.

Cell culture

Rat thyroid PCCL-3 cells were grown in Coon's modified Ham's F-12 medium. The medium was supplemented with 5% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), and a mixture of TSH (5 mU/ml), insulin (10 μg/ml), and hydrocortisone (3.2 ng/ml). When cells were 80% confluent, the medium was changed to TSH-deprived media, and cells were TSH starved for 3 d and then submitted to the different experimental conditions. PCCL-3 cells were either treated with TSH at 5 mU/ml for different times (1–15 min) or treated with 5 mU/ml TSH, 50 μm, or 1 μm PMA for 3 min. In some experiments, cells were pretreated with either the PKA inhibitor H-89 (0.5 μm), or the protein kinase C (PKC) inhibitor BIS (10 μm) for 30 min before incubation in the presence or absence of TSH. Cells were then placed on ice and processed as outlined below. For the immunofluorescence experiments, PCCL-3 cells were seeded in Poly-D-Lysine cellware two-well culture slides at approximately 5000 cells/well on d 0, TSH starved on d 1, and treated and processed for immunofluorescence on d 4.

TSA-201 cells, a clone of human embryonic kidney 293 cells, were maintained in DMEM containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). Rat kangaroo kidney epithelium (PtK2) cells were cultured in MEM (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and 0.1 mm nonessential amino acids.

Pendrin antibodies

A polyclonal primary IgY antibody against the carboxyl-terminal epitope ETELTEEELDVQDEAMRT of pendrin was raised in chicken (Aves Labs) (24). A polyclonal antibody directed against the extracellular epitopes of pendrin using genetic immunization with SLC26A4 cDNA was generated in rabbits (Genovac, Freiburg, Germany).

Biotinylation of cell surface proteins

Cells grown in 60-mm dishes were exposed to experimental conditions, placed on ice, washed three times with ice-cold PBS, and surface proteins were labeled for 20 min using 1 mg/ml EZ-link NHS-SS-biotin (Pierce Chemical Co., Rockford, IL) as described elsewhere (30–32). After labeling, cells were rinsed three times with PBS containing 100 mm glycine to quench unreacted biotin. Cells were lysed in modified radioimmunoprecipitation buffer [50 mm Tris-HCl (pH 8), 150 mm NaCl, 1% Nonidet P-40, and 1% sodium deoxycholate] containing protease inhibitors [cOmplete, Mini, EDTA-free Protein Inhibitor Cocktail Tablets (Roche Diagnostics, Mannheim, Germany) and 1 mm phenylmethylsulfonyl fluoride]. Proteins were quantified by Bradford assay (Bio-Rad, Hercules, CA), and 400–500 μg of proteins were incubated overnight in the presence of immobilized streptavidin (Pierce Chemical Co.). Beads were thoroughly washed and then resuspended in Laemmli's sample buffer solution, boiled for 5 min, and then analyzed by Western blotting.

Preparation of endosomes

PCCL-3 cells were treated with or without 5 mU/ml TSH for 5 min. The cells were placed on ice to terminate the incubation and were then washed three times with ice-cold PBS. Cells were resuspended in cold homogenization buffer containing 250 mm sucrose and 3 mm imidazole and protease inhibitors as above. After homogenization and brief centrifugation, the endosomes were fractionated on a sucrose flotation gradient as described (33).

Immunoprecipitation

Equal amount of proteins (400–500 μg) were incubated overnight with 25 μg (35 μl) of chicken pendrin antibody and 100 μl of agarose-conjugated goat antichicken IgY slurry (PrecipHen; Aves Labs). Immunoprecipitates were washed three times with PBS (5 min each wash), resuspended in Laemmli's sample buffer solution, boiled for 5 min, and then analyzed by Western blotting against PKA substrate (RRXS*/T*). Membranes were stripped and reblotted against pendrin as specified above.

Western blot analysis

Equal amounts of protein were resolved by 8% SDS-PAGE and analyzed by immunoblotting with a specific polyclonal chicken IgY antipendrin antibody (24). HRP-labeled goat antichicken IgY was used as secondary antibody. To determine the amount of phosphorylated pendrin, equal amounts of immunoprecipitates were resolved by 10% SDS-PAGE and analyzed by immunoblotting using a polyclonal phospho-PKA substrate antibody. HRP-labeled goat antirabbit antibody was used as secondary antibody. Membranes were stripped and reprobed against pendrin as described above. Densitometric analysis was performed using the ImageJ program from the National Institutes of Health (http://rsb.info.nih.gov/ij/).

Immunofluorescence and confocal microscopy

PCCL-3 cells were seeded on d 0 in Poly-D-Lysine cellware two-well culture slides (∼5 × 103 cells/well). Cells were TSH starved on d 1 and treated with or without 5 mU/ml TSH for 5 min on d 4. After treatment, cells were placed on ice and washed three times with ice-cold PBS, then fixed at room temperature with 1% glutaraldehyde in PBS. When indicated, cells were permeabilized with 0.2% Triton for 15 min.

Determination of intracellular iodide

PCCL-3 or TSA cells were grown to 75–80% confluency. PCCL-3 cells were then TSH-deprived for 3 d. TSA cells were incubated in the absence or in the presence of FSK (50 μm) as indicated. For the iodide assays, the cells were incubated in Hanks' balanced salt solution supplemented with 10 mm HEPES (pH 7.4), 1 mm dithiothreitol, 1 mm methimazole, and 10−5 m cold NaI labeled with Na125I (20 mCi/mmol) for 30 min. Subsequently, 1 mm perchlorate was added for 10 min to block further iodide uptake by NIS. This was followed by treatment of the cells with TSH (5 mU/ml) for either 0, 1, 3, 5, or 10 min. To block pendrin-mediated iodide efflux, the cells were concomitantly incubated with the antibody raised by genetic immunization and recognizing conformational extracellular epitopes (dilution 1:1000). A polyclonal, E-cadherin antibody was used in a dilution of 1:1000 to determine whether the observed inhibition of pendrin-mediated iodide efflux is specific or not. Supernatatants were aspirated and the cells were lysed using 1% Triton in PBS. The intracellular iodide content was determined by measuring radiolabeled iodide in the cell lysates using a Gamma Counter as previously described (24).

Live cell imaging

PtK2 cells transfected with enhanced green fluorescent protein (GFP)-tagged wild-type pendrin or the mutant eliminating the putative phosphorylation site at position 717 (T717A) were imaged using an Olympus DSU spinning disk confocal fitted on an Olympus IX-81 microscope (Olympus, Center Valley, PA) enclosed in a 37 C-heated CO2 chamber in the Northwestern University Cell Imaging Facility. Cells were examined under a ×60 oil-immersion objective. Images were acquired at a 100-msec exposure every 5 min. After 1 h, FSK (50 μm) was added to the cells, and images were collected for another hour. To image cells in the absence of CO2, the cell medium was mixed with equal volumes of Leibovitz L-15 medium without Phenol Red (Invitrogen Life Technologies, Inc.) supplemented with 10% FBS. For long-term live imaging, focal plane was actively maintained by the zero drift compensation mechanism of the Olympus IX-81 microscope.

Statistical analysis

All analyses were performed in at least three independent experiments. Data are reported as means ± sem. Statistical analysis was performed by one-way ANOVA, using Tukey's correction. When only two groups were compared, analysis was performed using Student's t test. Results were considered significant when P < 0.05.

Results

TSH rapidly regulates pendrin abundance at the plasma membrane from intracellular pools

Immunoblot experiments

Cell surface labeling of plasma membrane proteins with biotin and analysis by immunoblotting against pendrin after pull down with streptavidin demonstrates that TSH rapidly increases the amount of pendrin at the plasma membrane (Fig. 1A). In contrast, the amount of total protein does not change (Fig. 1B).

Fig. 1.

Fig. 1.

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TSH rapidly regulates pendrin abundance at the plasma membrane. PCCL-3 cells treated for different time points with 5 mU/ml were surface labeled with biotin, and membrane proteins were pulled down with streptavidin. Immunoblots were then performed with an antibody against a carboxy-terminal domain of pendrin. The results reveal that TSH rapidly increases the amount of pendrin at the plasma membrane (Panel A). In contrast, the amount of total amount of pendrin, as analyzed by Western blotting of total cell lysates, does not change (Panel B). Immunoblot analysis of endosomal proteins fractionated on a sucrose flotation gradient demonstrate a decrease in pendrin abundance in response to incubation with 5 mU/ml TSH for 5 min (Panel C). The findings suggest that pendrin is translocated from endosomal compartments to the plasma membrane. C, Control. Means ± sem; **, P ≤ 0.01 and representative immunoblot. All analyses were performed in at least three independent experiments.

Analysis of endosomes fractionated on a sucrose flotation gradient by immunoblotting against pendrin reveals a decrease in pendrin abundance in response to incubation with TSH for 5 min, suggesting a translocation from endosomal compartments to the plasma membrane (Fig. 1C).

Immunofluorescence experiments

Confocal microscopy using an antibody generated by genetic immunization and recognizing the extracellular domains of pendrin demonstrates that TSH rapidly increases pendrin abundance at the plasma membrane in nonpermeabilized PCCL-3 cells (Fig. 2, left panels). Permeabilization of PCCL-3 cells before immunofluorescence allows the visualization of pendrin in intracellular compartments. PCCL-3 cells incubated in absence of TSH showed mostly perinuclear localization of pendrin (Fig. 2, right panels). After treatment with TSH for 5 min, the pattern of fluorescence changes, and the amount of pendrin at the plasma membrane is more pronounced (Fig. 2, right panels), findings that are consistent with the immunoblot data.

Fig. 2.

Fig. 2.

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Immunofluorescence (IF) with a pendrin antibody recognizing extracellular epitopes. Immunofluorescence was performed with an antibody developed by genetic immunization and thus recognizing conformational extracellular domains of pendrin without (left panels) or with previous permeabilization (right panels). The cells were then analyzed by confocal microscopy. The results demonstrate that TSH rapidly increases pendrin abundance at the plasma membrane in nonpermeabilized PCCL-3 cells (left panels). Permeabilization of PCCL-3 cells before immunofluorescence allows the visualization of pendrin in intracellular compartments (right panels). Permeabilized PCCL-3 cells incubated in absence of TSH showed mostly perinuclear localization of pendrin. After treatment with 5 mU/ml TSH for 5 min, the pattern of fluorescence changes and the amount of pendrin at the plasma membrane were more pronounced. Consistent with the immunoblot results (Fig. 1), these findings demonstrate a rapid translocation of pendrin from intracellular compartments to the membrane in response to TSH. DAPI, 4′,6-diamidino-2-phenylindole.

TSH regulates pendrin membrane abundance via a PKA-dependent pathway

To determine whether the TSH-mediated regulation of pendrin occurs through the PKA or the PKC signaling cascade, the pathways were blocked with H-89 (PKA) or BIS (PKC). The PKA inhibitor H-89 blocks the TSH-induced increase in pendrin abundance at the plasma membrane, whereas the PKC inhibitor BIS does not (Fig. 3A). In addition, the adenylyl cyclase agonist FSK mimics the TSH-induced increase in pendrin abundance at the plasma membrane (Fig. 3B), whereas cells incubated in the presence of the PKC agonist PMA do not show any increase in pendrin membrane abundance compared with controls (data not shown). Although pendrin migrated typically as a single band, the occasional appearance of two separate bands probably reflects differences in secondary modifications (Fig. 3B, lane 2).

Fig. 3.

Fig. 3.

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TSH regulates pendrin membrane abundance via a PKA-dependent pathway. To determine whether the TSH-mediated translocation of pendrin occurs through the PKA or the PKC signaling cascade, the pathways were blocked with H-89 (PKA) or BIS (PKC). The PKA inhibitor H-89 blocks the TSH-mediated insertion of pendrin into the plasma membrane, whereas the PKC inhibitor BIS does not have an effect (Panel A). The adenylyl cyclase agonist FSK mimics the TSH-induced increase in pendrin abundance at the plasma membrane (Panel B). Cells incubated in the presence of the PKC agonist PMA do not show an increase in pendrin membrane abundance compared with controls (data not shown). C, Control. Means ± sem; *, P ≤ 0.05; **, P ≤ 0.01, and representative immunoblots. All analyses were performed in at least three independent experiments.

TSH and FSK phosphorylate pendrin

To assess whether exposure to TSH results in phosphorylation of pendrin, pendrin was immunopreciptiated with a polyclonal antibody raised against the carboxy-terminal domain. After transfer, immunoblots were performed with an antibody specifically recognizing PKA-phosphorylated substrates. The membranes were then stripped and probed with the antipendrin antibody. The amount of phosporylated protein was normalized to the total amount of pendrin. The results suggest that both TSH and FSK phosphorylate pendrin at a PKA consensus site (Fig. 4).

Fig. 4.

Fig. 4.

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Phosphorylation of pendrin by TSH and FSK. To assess whether exposure to TSH results in phosphorylation of pendrin, pendrin was immunopreciptiated with a polyclonal antibody raised against a carboxy-terminal domain. Immunoblots were performed with an antibody specifically recognizing PKA-phosphorylated substrates (P-PKA), and the membranes were then reprobed with a pendrin antibody. PKA-phosphorylated substrate was normalized for the detected amount of pendrin. The results demonstrate that both TSH and FSK phosphorylate pendrin. C, Control. Means ± sem; *, P ≤ 0.05. and representative immunoblots. All analyses were performed in at least three independent experiments.

TSH rapidly increases iodide efflux in PCCL-3 rat thyroid cells

To assess the effect of TSH on intracellular iodide accumulation in thyroid cells, TSH-deprived PCCL-3 cells were incubated with radioactive iodide for 30 min to allow for iodide uptake to reach a steady state as described (4). Cells were then incubated without or with 1 mm sodium perchlorate to inhibit iodide uptake by NIS, followed by incubation in the presence or absence of TSH for different time intervals. As shown in Fig. 5, TSH induces a rapid decrease in intracellular iodide content in the presence of perchlorate, a finding that is consistent with the rapid increase in iodide efflux observed in polarized primary thyrocytes and FRTL-5 cells (11, 12). In the absence of perchlorate as competitive inhibitor of NIS, the addition of TSH leads to an increased iodide uptake. Because PCCL-3 cells (and the Fisher rat cell line FRTL-5) are poorly polarized, they are not suitable for further characterization in bicameral systems.

Fig. 5.

Fig. 5.

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TSH rapidly increases iodide efflux in PCCL-3 rat thyroid cells. To assess the effect of TSH on intracellular iodide accumulation, PCCL-3 cells deprived of TSH for 3 d were incubated with radioactive iodide for 30 min to allow for iodide uptake to reach a steady state as described. Cells were then incubated in the absence or presence of 1 mm sodium perchlorate, which inhibits iodide uptake by NIS, followed by incubation in the presence or absence of TSH for different times. In the absence of perchlorate, the addition of TSH leads to an increased iodide uptake, suggesting an increased NIS activity. In contrast, TSH induces a rapid decrease in intracellular iodide content in the presence of perchlorate, a finding that is consistent with the rapid increase in iodide efflux observed in polarized primary thyrocytes and FRTL-5 cells (11, 12). C, Control. Means ± sem; *, P ≤ 0.05. All analyses were performed in at least three independent experiments.

To assess whether the observed TSH-induced efflux is indeed mediated by pendrin, PCCL-3 cells were incubated with an antibody raised by genetic immunization and thus recognizing conformational extracellular epitopes. Pretreatment with this antibody increases the content of intracellular iodide, indicating that the efflux of iodide has decreased through specific blockade of pendrin (Fig. 6, lane 2). In contrast, incubation with an E-cadherin antibody does not have any impact on iodide content of the cells (lane 3), as indicated by an unchanged intracellular iodide content compared with untreated controls (lane 1). Using this pendrin antibody, the TSH-induced iodide efflux can be blocked (lane 4, 5), but this is not possible with an unrelated antibody (lane 7).

Fig. 6.

Fig. 6.

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Effect of a specific pendrin antibody on iodide efflux. To block pendrin-mediated iodide efflux, PCCL-3 cells were incubated with an antibody raised by genetic immunization and recognizing conformational extracellular epitopes (POS Ab). Pretreatment with this antibody increases the content of intracellular iodide, indicating that the efflux of iodide has decreased through specific blockade of pendrin (lane 2). In contrast, incubation with an unrelated E-cadherin antibody (lane 3) (CAD Ab) did not have any influence on iodide efflux as indicated by an unchanged intracellular iodide content compared with untreated controls (lane 1). The TSH-induced iodide efflux (lane 4) can be blocked by the specific pendrin antibody (lane 5) but not with an E-cadherin antibody (lane 7). C, Control. Means ± sem; *, P ≤ 0.05; **, P ≤ 0.01. All analyses were performed in at least three independent experiments.

To characterize the potential functional role of the putative PKA phosphorylation site at T717, iodide efflux mediated by wild-type pendrin and the mutant T717A were directly compared. TSA cells were incubated in iodide-containing uptake buffer in the absence or presence of FSK for 30 min. Subsequently, 1 mm perchlorate was added for 10 min to block further iodide uptake by NIS before determining the intracellular iodide concentration. TSA cells transiently expressing NIS alone demonstrate a 91% increase in iodide uptake in the absence of FSK, compared with control cells transfected with the empty vector (Fig. 7). In the absence of perchlorate, FSK induces a further increase in intracellular iodide concentration (data not shown), which is not present if the cells are treated with perchlorate for 10 min. The intracellular iodide amount is reduced by 50% in cells cotransfected with NIS and pendrin in the absence of FSK, whereas concomitant incubation with FSK leads to a 70% decrease (P < 0.001) (Fig. 7). Cotransfection of a pendrin mutant containing the modified PKA phosphorylation site (PDS T717A) and NIS led to a 33% decrease in intracellular iodide demonstrating that the mutant is less efficient in mediating iodide efflux but that it retains partial function (NIS/PDS vs. NIS/T717A, P < 0.001). In the presence of FSK, there is no significant decrease in intracellular iodide concentration (P > 0.05), suggesting that the mutant does not respond to stimulation of the cAMP pathway (Fig. 7).

Fig. 7.

Fig. 7.

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Intracellular iodide content of TSA cells expressing wild-type and mutant pendrin proteins. To characterize the potential functional role of the putative PKA phosphorylation site at T717, iodide efflux mediated by wild-type pendrin and the mutant T717A were directly compared. TSA cells were incubated in iodide-containing uptake buffer in the absence or presence of FSK for 30 min. Subsequently, 1 mm perchlorate was added for 10 min to block further iodide uptake by NIS before determining the intracellular iodide concentration. TSA cells transiently expressing NIS alone demonstrate a 91% increase in iodide uptake in the absence of FSK, compared with control cells transfected with the empty vector. The intracellular iodide amount is reduced by 50% in cells cotransfected with NIS and pendrin in the absence of FSK, whereas concomitant incubation with FSK leads to a 70% decrease (P < 0.001). Cotransfection of a pendrin mutant containing the modified PKA phosphorylation site (PDS T717A) and NIS led to a 33% decrease in intracellular iodide, demonstrating that the mutant is less efficient in mediating iodide efflux, but that it retains partial function (NIS/PDS vs. NIS/T717A, P < 0.001). In the presence of FSK, the intracellular iodide content does not decrease significantly (P > 0.05), suggesting an impaired response of the mutant to stimulation of the cAMP pathway. Data shown are representative of more than three independent experiments. Values are the means of triplicates ± se.

Live imaging

To assess the effects of the T717A PKA mutant, we expressed wild-type and mutant GFP-tagged pendrin in PtK2 cells and analyzed the localization using time-lapse imaging of living cells. In the absence of FSK, the majority of the GFP-tagged wild-type pendrin is located in intracellular compartments (Fig. 8). Within 15 min after addition of FSK, a significant amount of wild-type pendrin translocates to the plasma membrane. The majority of the putative PKA phosphorylation site mutant (T717A) is located in the cytoplasm, but a small portion of the protein is present at the plasma membrane (Fig. 8). In response to FSK, there is only a very modest increase in the amount of pendrin at the plasma membrane.

Fig. 8.

Fig. 8.

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Subcellular distribution of the GFP-tagged wild-type pendrin in living PtK2 cells. PtK2 cells transiently expressing GFP-tagged wild-type pendrin were imaged using time-lapse confocal microscopy. In the absence of FSK, the majority of wild-type pendrin is retained in intracellular compartments. Within 15 min after adding FSK, a significant amount of the protein translocates to the plasma membrane (shown by arrows). This pattern is observed for 1 h after treatment with FSK. These results indicate that plasma membrane targeting of pendrin is mediated, in part, by the PKA pathway, consistent with previous findings obtained in FRTL-5 cells (4). The majority of the putative PKA phosphorylation site mutant (T717A) is located in the cytoplasm, but a small portion of the protein is present at the plasma membrane. In response to FSK, there is only a very modest increase in the amount of pendrin at the plasma membrane.

Discussion

First evidence that apical iodide transport in thyroid cells is a regulated process was provided by demonstrating that iodide accumulates in the cytoplasm before reaching the follicle (6). Subsequent studies in animal models and cultured thyroid cells revealed that iodide efflux to the follicular lumen is rapidly accelerated by TSH (1, 4, 8, 11, 12). These data, and electrophysiological studies performed with inverted plasma membrane vesicles (34), suggested the existence of apical iodide transporters, but the identity of this or these channel(s) has not been elucidated. The effects of TSH on pendrin expression in thyroid cell lines are, in part, conflicting (21, 35, 36). In FRTL-5 cells, one study reported no induction of SLC26A4 mRNA determined by Northern blot analyses after treatment with low doses of TSH (21), whereas others reported increased mRNA expression measured by RT-PCR after exposure to TSH and FSK (35). In PCCL-3 cells, high doses of TSH, FSK, and 8-Bromo-cAMP seem to increase pendrin mRNA and protein expression (36).

However, it is well established that TSH stimulates apical iodide efflux (1, 4, 8, 11, 12), and it has a rapid effect on the exit rate of thyroidal iodide in rats (1). Subsequent studies have shown that this is a regulated process (2, 6), which occurs specifically at the apical membrane of thyroid follicular cells (8). TSH stimulates iodide efflux in FRTL-5 cells (4, 8, 11, 12), which, as PCCL-3 cells, lack distinct polarity, as well as in polarized thyroid cells (4, 8, 11, 12). This initial effect of TSH on iodide transport occurs very rapidly and facilitates the transport of iodide into the lumen (4, 8, 11, 12). Nilsson et al. (8), studying iodide efflux in polarized porcine thyroid cells cultured in bicameral chambers, demonstrated that TSH-mediated iodide efflux occurs selectively at the apical membrane. The increase in iodide efflux was evident after 2–4 min, peaked at 4–6 min, and subsequently declined (8). Similarly, Weiss et al. (12) reported that the addition of TSH to FRTL-5 cells leads to a 20–30% decrease in the amount of intracellular iodide after 5 min, and this persists for at least 20 min.

The clinical phenotype of patients with PDS (goiter, impaired iodide organification) (15), the apical location of pendrin in follicular cells (21), and functional data demonstrating iodide transport in Xenopus oocytes (20), heterologous unpolarized cell lines (22–24, 37), as well as polarized cells (24), suggested a possible role for pendrin as an apical iodide channel in the thyroid. This concept has, however, been questioned (7). The main reasons brought forward against a physiological role of pendrin in apical iodide transport include that: 1) targeted disruption of the Slc26a4 gene in mice does not result in an overt thyroid phenotype (38), even after exposure to an iodine-deficient diet (28, 29); 2) pendrin has a distinct role in the kidney as chloride/bicarbonate exchanger (25, 39); 3) TSH does not have a major impact on SLC26A4 gene expression (as outlined above, the reported findings are, however, controversial) (21, 35, 36); 4) all functional data have been derived in heterologous cells; and 5) that pendrin has to display a substantial iodide selectivity, because the cytoplasmic and luminal chloride concentrations are higher than the iodide concentrations (7). Importantly, recent studies in Xenopus oocytes have not only formally demonstrated that SLC26A4 functions as a coupled, electroneutral iodide/chloride, iodide/bicarbonate, and chloride/bicarbonate exchanger with a 1:1 stoichiometry (40) but also shown that SLC26A4 preferentially transports iodide even in the presence of high chloride concentrations (40). Moreover, SLC26A4 has been shown to mediate apical iodide secretion into parotis ducts (40).

The results of this study demonstrate that TSH rapidly increases pendrin abundance at the plasma membrane (Figs. 1 and 2). This is associated with a rapid decrease of pendrin in endosomal compartments, suggesting that TSH increases translocation of pendrin to the plasma membrane or that it decreases its endocytosis (Figs. 1C and 2). The increase in pendrin abundance at the plasma membrane appears to be mediated by the PKA pathway (Fig. 3). Moreover, exposure of thyroid cells to TSH and FSK result in the phosphorylation of pendrin (Fig. 4), an observation that suggests that translocation to the membrane may be dependent on phosphorylation. Pendrin contains a PKA phosphorylation motif in its intracellular carboxy terminus. As shown in Fig. 7, incubation of cells cotransfected with NIS and pendrin with FSK leads to a significant decrease in intracellular iodide content. In contrast, cells contransfected with NIS and a mutation of the putative phosphorylation site T717A show a decreased ability to mediate iodide efflux, indicating that this mutation leads to an impaired function compared with the wild type. Moreover, T717A is not able to mediate a significant increase in iodide efflux in response to FSK. Consistent with these functional data, imaging studies with T717A fused to the GFP show that only a small portion of the protein is present at the plasma membrane and that it barely increases after treatment with FSK (Fig. 8).

The functional data shown here are of particular relevance (Figs. 5 and 6). Remarkably, the time course of the observed increase in membrane abundance of pendrin (Fig. 1) correlates well with the increase in iodide efflux reported in earlier studies in FTRL-5 or primary thyroid cells (1, 8, 11, 13), and it can easily be reproduced in PCCL-3 cells (Fig. 5). Although this temporal correlation could suggest that the increase of iodide efflux is due to an increase in the abundance of pendrin in the membrane, it does not prove a causal relationship. However, using a pendrin antibody generated by genetic immunization and recognizing extracellular conformational epitopes, we were able to specifically inhibit iodide efflux in the presence or absence of TSH (Fig. 6). This observation suggests that pendrin can mediate iodide efflux from thyroid cells in vitro. However, the exact physiological role of pendrin in the thyroid needs to be further clarified for several reasons. Firstly, it is obvious that apical iodide efflux cannot be solely dependent on pendrin, because patients with PDS only have a partial organification defect, and under conditions of normal or high iodide intake, there is typically no obvious thyroid phenotype (41, 42). This emphasizes that apical iodide efflux can occur through a distinct iodide transporter or through chloride channels. Targeted disruption of the chloride channel 5 in mice is associated with a pendrin-like phenotype (43), and in thyroid tissue from a patient with PDS, chloride channel 5 expression was increased leading to the speculation that this may result in compensatory apical iodide efflux through this channel (44). It should also be noted, that the transepithelial potential and pH of thyroid follicles is reduced in Slc26a4−/− mice, suggesting that pendrin mediates influx of chloride and efflux of bicarbonate (45). Thus, an alternative explanation for the positive perchlorate test could perhaps result from a change in the intrafollicular pH, which could, for example, alter the efficiency of thyroid peroxidase.

In conclusion, the data presented here provide evidence that TSH regulates pendrin by posttranscriptional mechanisms in rat thyroid cells through a PKA-dependent pathway. The rapid translocation of pendrin to the plasma membrane results in an increase in iodide efflux. Although apical iodide efflux can be mediated by at least one other iodide transporting entity, the findings suggest that pendrin may have a physiological role in mediating iodide efflux into the follicular lumen, a key step in the biosynthesis of thyroid hormones (5).

Acknowledgments

We thank Marissa Michaels, B.S., for valuable technical assistance and Juan Carlos Solis, M.D., Ph.D., for insightful discussions.

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Grant 1R01DK63024-01 (to P.K.), the National Heart, Lung, and Blood Institute Grant K01HL080966-01 (to A.P.C.), the Endocrine Fellows Foundation Grant 98000003 (to L.P. and P.K.), and a gift from Mr. David Wiener (P.K.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
BIS
Bisindolylmaleimide I
FBS
fetal bovine serum
FSK
forskolin
GFP
green fluorescent protein
HRP
horseradish peroxidase
NIS
sodium-iodide symporter
PDS
Pendred syndrome
PKA
protein kinase A
PKC
protein kinase C
PMA
phorbol 12-myristate 13-acetate.

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