European Journal of Endocrinology (2001) 145 591±597 ISSN 0804-4643

CLINICAL STUDY Sodium/iodide (NIS) and are expressed differently in hot and cold nodules of toxic multinodular goiter Diego Russo, Stefania Bulotta, Rocco Bruno2, Franco Arturi1, Paolo Giannasio2, Michael Derwahl5, Jean-Michel Bidart3, Martin Schlumberger4 and Sebastiano Filetti1 Dipartimento di Scienze Farmacobiologiche and 1Dipartimento di Medicina Sperimentale e Clinica, UniversitaÁ di Catanzaro, 88100 Catanzaro, 2Ospedale Civile di Tinchi-Pisticci, Matera, Italy, 3Departments of Clinical Biology and 4Nuclear Medicine, Institut Gustave-Roussy, 94805 Villejuif, France and 5Department of Medicine, Division of Endocrinology, St Hedwig Hospital and Humboldt University, 10115 Berlin, Germany (Correspondence should be addressed to Sebastiano Filetti, Dipartimento di Medicina Sperimentale e Clinica, UniversitaÁ degli Studi di Catanzaro, Via T. Campanella, 115, 88100 Catanzaro, Italy; Email: ®[email protected])

Abstract Objective: The expression of two iodide transporters, the sodium/iodide symporter (NIS) and pendrin, was analyzed in thyroid tissues of patients with toxic multinodular goiter (TMNG) and non-toxic multinodular goiter (MNG). Methods: The levels of NIS and pendrin were analyzed in total extracts from nodular and non-nodular tissues by Western blot. Results: In tissue samples from TMNG, we found an increased expression of NIS (2.5-fold) in the hot nodules, and similar levels between cold nodules and non-nodular tissues. In contrast, the levels of pendrin were slightly increased in both hot and cold nodules from TMNG, and decreased (about twofold) in cold nodules from MNG. We also noticed that there was no relationship between NIS and pendrin expression. Conclusions: Our data demonstrate that hot nodules from TMNG express a higher number of iodide transporters (mainly NIS), whereas cold nodules from TMNG, but not from MNG, show levels of the two proteins comparable with normal tissue, suggesting a role in vivo of TSH in maintaining the expression of NIS and pendrin protein in normal thyroid tissue. Finally, different mechanisms are involved in the regulation of NIS and pendrin expression.

European Journal of Endocrinology 145 591±597

Introduction For years, thyroid nodules have been characterized on the basis of their ability to concentrate radioiodine, Iodide uptake is the ®rst step in thyroid hormone thus considered functioning or non-functioning at 131I- production by thyroid follicles. Thyroidal iodide trap- scintiscan and named `hot' or `cold' respectively. Many ping and concentration from blood are achieved by an studies have shown abnormal expression of the NIS active, energy-dependent transport process across the in various thyroid disorders (reviewed in 10, 11), baso-lateral plasma membrane of the thyrocytes; iodide especially in thyroid malignancies. In contrast, few data is then transported trans-cellularly to the apical are available on PDS gene expression (8), or on the membrane, where it is organi®ed (1). The way to relationship between NIS and pendrin expression at the clarify the molecular mechanisms underlying iodide protein level (6±9, 12). transport in thyroid cells has been opened up as a result In this study, using Western blot analysis we of the cloning and expression studies of two proteins examined the expression of NIS and pendrin proteins mainly involved in this process, namely the sodium/ in a series of 25 thyroid nodules from ten unrelated iodide symporter (NIS) (2, 3) and pendrin (4, 5). NIS is patients with toxic multinodular goiter (TMNG), located in the baso-lateral plasma membrane of including both hot and cold nodules, in order to clarify thyrocytes (6, 7) and catalyzes active I2 uptake into the role of these two iodide transporters in the the cell, while pendrin, discovered as the defective functional behavior of the nodular lesions. Indeed, the protein in (PDS) (4), is a chloride/ simultaneous presence of hot and cold nodules in the iodide transporter expressed in the apical membrane (8, same patient makes this disease a good model for 9), presumably responsible for the I2 accumulation in excluding interference due to genetic and environmen- the colloid. tal factors. For the ®rst time, the protein expression q 2001 Society of the European Journal of Endocrinology Online version via http://www.eje.org

Downloaded from Bioscientifica.com at 10/02/2021 04:05:42AM via free access 592 D Russo and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2001) 145 levels were compared with the normal, non-nodular another ®ve patients with MNG and non-nodular tissue of each patient. To evaluate the in¯uence of tissues from two patients with Graves' disease were thyroid-stimulating hormone (TSH), which is obviously also examined. Non-nodular, normal tissue from each suppressed in patients with TMNG, ®ve cold nodules patient was also investigated. All patients lived in an from patients with non-toxic multinodular goiter (MNG) area of moderate±severe iodine de®ciency. Informed who had detectable levels of TSH were also investigated. consent was obtained from all patients. A map of the nodules, as they appeared at scintiscan and ultrasound Materials and methods examination, was provided for the surgeon, so that nodules de®ned by scintiscan could be unequivocally Materials identi®ed post-operatively. Tissue specimens, obtained at the time of surgery, were frozen in liquid nitrogen. HEPES, sucrose, dithiothreitol (DTT), EDTA, phenyl- Toxic multinodular goiter was diagnosed on the basis of methylsulfonyl ¯uoride (PMSF), aprotinin, leupeptin clinical thyrotoxicosis, elevated serum levels of free and anti-human beta-actin monoclonal antibody were , and undetectable TSH levels, asso- obtained from Sigma-Aldrich S.r.l. (Milan, Italy); ciated with a multinodular goiter presenting predomi- nitrocellulose membranes, horseradish peroxidase con- nant 131I uptake in one (or more) nodule(s) as shown jugated anti-rabbit or anti-mouse antibodies, auto- by 131I scanning. The thyroid nodules were classi®ed as radiography ®lm and enhanced chemiluminescence hot or cold according to the scintigraphic examination. (ECL-Plus) Western blotting detection reagents were All nodules were histologically classi®ed as adenomas from Amersham Pharmacia biotech (Milan, Italy); according to the WHO recommendations (13). The protein assay reagent, non-fat dry milk and molecular clinical features of the patients are shown in Table 1. weight markers were from Bio-Rad Laboratories This study was approved by the local ethical committee. (Segrate, Milan, Italy). Protein extraction and Western blot Patients Total proteins were extracted from thyroid tissues as A total of 25 nodules from ten unrelated patients with follows. Frozen tissue was thawed and homogenized in thyroid TMNG were studied. Five nodular tissues from 1 ml of buffer containing 250 mmol/l sucrose,

Table 1 Clinical features of the patients with TMNG. Hormonal values were obtained at the time of diagnosis, when 131I scintiscan was performed. The patients were then treated with anti-thyroid drugs and operated on when a state of subclinical hyperthyroidism was reached. Normal values: TSH: 0.35±5.5; FT3 2.3±4.2; FT4: 0.8±1.5.

131 No. Sex/Age TSH (mIU/l) FT3 (pg/ml) FT4 (ng/dl) Nodules Diameter (mm) Ultra-sonography I scintiscan

1 F/51 0.01 4.38 1.5 1a 20 Mixed Cold 1b 25 Mixed Hot 1c 18 Mixed Hot 2 F/65 0.01 4.65 1.76 2a 15 Mixed Cold 2b 18 Mixed Cold 2c 20 Mixed Hot 3 F/60 0.01 4.1 1.2 3a 16 Mixed Cold 3b 18 Mixed Hot 4 F/81 0.1 3.51 1.01 4a 13 Mixed Cold 4b 13 Hypoechoic Hot 5 M/62 0.01 3.92 1.05 5a 70 Hypoechoic Cold 5b 27 Hypoechoic Hot 6 M/71 0.01 3.58 0.8 6a 20 Isoechoic Cold 6b 15 Mixed Cold 6c 30 Hypoechoic Cold 6d 50 Isoechoic Hot 7 F/71 0.38 7.15 1.23 7a 16 Isoechoic Cold 7b 17 Mixed Hot 7c 17 Mixed Hot 8 M/75 1.8* 3.78* 0.8* 8a 50 Mixed Cold 8b 25 Mixed Hot 9 F/54 0.01 5.40 21.2 9a 24 Hypoechoic Cold 9b 56 Hypoechoic Hot 10 F/43 0.07 9.0 3.04 10a 40 Mixed Cold 10b 40 Isoechoic Hot

*Values obtained during treatment with anti-thyroid drugs. Almost all patients showed only a T3 thyrotoxicosis, as expected in an area of severe iodine de®ciency. FT3, free tri-iodothyronine; FT4, free thyroxine. www.eje.org

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10 mmol/l HEPES-KOH (pH 7.5), 1 mmol/l EDTA, 1 mmol/l PMSF, 10 mg/ml leupeptin, 10 mg/ml apro- tinin. The homogenate was centrifuged at 14 000 g (4 8C for 15 min) and the supernatant (which con- tained the whole cell lysate) was quanti®ed spectro- photometrically using the Bradford method. Fifty micrograms of protein were loaded onto a 4±20% gradient SDS polyacrylamide gel and subjected to electrophoresis at a constant voltage (120 V). Figure 1 Western blot analysis under reduced conditions of protein Electroblotting to a Hybond-P ECL nitrocellulose extracts from tissues of two patients with Graves' disease, NIH 3T3 membrane was performed for 2 h at 125 mA using a ®broblasts and normal and nodular tissues from two patients, using Mini Trans blot electroblotting system. Blocking was a polyclonal anti-NIS antibody (upper panel) and a monoclonal done using TTBS/milk (TBS, 1% Tween 20 and 5% anti-human beta-actin antibody (lower panel) (see Materials and methods). A representative of three separate experiments is non-fat dry milk) for 2 h at room temperature. The shown. Lanes 1 and 2=positive control (Graves' tissue); lane membrane was then incubated with a 1/1000 dilution 3=negative control (®broblasts); lanes 4, 5, 6=non-nodular of af®nity puri®ed rabbit anti-NIS (6) or 1/1500 (normal), hypofunctioning (cold) nodule and hyperfunctioning (hot) dilution of anti-pendrin (8) polyclonal antibody or a nodule from a patient with TMNG (no. 4 in Table 1); lanes 7, 8, 1/5000 dilution of mouse monoclonal anti-human 9=non-nodular (normal), hypofunctioning (cold) nodule and hyperfunctioning (hot) nodule from a patient with TMNG (no. 5 in beta-actin antibody, overnight at 4 8C in TTBS/milk. Table 1). After one 15-min and two 5-min washes in TTBS, the membrane was incubated with a 1/2000 dilution of a horseradish peroxidase conjugated anti-rabbit or anti- mouse antibody in TTBS/milk. After one 15-min and NIS protein as a major band that migrated with the two 5-min washes in TTBS, the protein was visualized 78 kDa molecular weight marker (Fig. 1). Pre-incuba- by an enhanced chemiluminescence Western blot tion of the same antibody with 50 mg of peptide hNIS detection system. Quanti®cation was achieved by 615±643 (see Materials and methods) determined the densitometric scanning. The same procedure was disappearance of the signal, demonstrating the speci- repeated after pre-incubating the antibody overnight ®city of the band detected (data not shown). The levels at the same dilutions with 50 mg of peptide hNIS 615± of NIS protein were variable among hot nodules, but 643 (6), or hPDS 765±780 (8). The anti-NIS and anti- they were always higher than in the corresponding pendrin antibodies used in this study recognized NIS normal tissues, with an average increase of approxi- and pendrin proteins at the baso-lateral and apical mately 2.5-fold. In contrast, only slight and not thyrocyte plasma membrane respectively, and no signal signi®cant differences were detected between cold in the cytosol and/or nucleus was detectable in nodules and perinodular specimens of the same immunohistochemical analysis (6, 8). patients (Fig. 2 and Table 2).

Statistical methods The expression levels of NIS and pendrin detected in the different nodular tissues were compared with those in Detection of pendrin protein normal thyroid tissues. The signi®cance of differences between group means was analyzed by Student's By using a polyclonal anti-pendrin antibody (8) in (paired) t-test. A level of P , 0:05 was considered immunoblot experiments, a major band of approxi- statistically signi®cant. The relationships between NIS mately 95 kDa was detected in the protein extracts of and pendrin expression were studied using the Pearson thyroid tissues from patients with Graves' disease and correlation coef®cient. All calculated probability values TMNG (Fig. 3). Also in this case, the speci®city was were two-tailed and a P value of ,0.05 was considered demonstrated by the absence of the band when signi®cant. incubating the rabbit anti-PDS immune serum with an excess amount of hPDS 765±780 peptide (data not shown). Again, variability among samples was Results detected, with a slight increase of the pendrin in both cold and hot nodules as compared with the non- Detection of NIS protein nodular tissues (Fig. 2 and Table 2). Western blot analysis was carried out with protein Western blot analysis in cold nodules from patients extracts from hot and cold nodules and the perinodular with MNG and detectable levels of TSH showed a tissue of thyroid toxic multinodular goiter. In all the decrease in both NIS and pendrin expression as samples, and in the Graves' thyroid tissues used as compared with the normal tissues (Fig. 4 and positive controls, anti-NIS antibody did recognize the Table 2).

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Figure 2 Levels of NIS and pendrin protein in normal and pathological thyroid tissues. Western blot analyses were carried out with protein extracts from thyroid tissues using a polyclonal anti-NIS or anti-pendrin antibody. Values represent the NIS/beta-actin (A) and the pendrin/ beta-actin (B) protein ratio expressed as percentage of increment over the normal tissue of each patient in arbitrary units as measured by laser densitometry. Data from nodules of ten unrelated patients (numbered as in Table 1) are shown.

Table 2 NIS and pendrin protein levels in normal and nodular thyroid tissues of patients with TMNG and MNG. Values represent arbitrary units as measured by laser densitometry, indicating the mean^S.D. of the NIS or pendrin/beta-actin expression calculated as the ratio between the modular and the corresponding normal Figure 3 Western blot analysis under reduced conditions of protein tissue of each patient (taken as 100). extracts from a Graves' tissue (positive control), NIH 3T3 ®broblasts (negative control) and nodular (hot and cold) and NIS protein Pendrin protein non-nodular tissues from two patients with TMNG, using a polyclonal anti-pendrin antibody (upper panel) and a monoclonal TMNG anti-human beta-actin antibody (lower panel). A representative of Hot nodular tissues n 12 240.0^97.2* 155.4^59.1* three separate experiments is shown. Lane 1 negative control; ˆ † * lanes 2, 3, 4 non-nodular (normal), hypofunctioningˆ (cold) Cold nodular tissues n 13 116^36.7 170.0^46.4 ˆ MNG ˆ † nodule and hyperfunctioning (hot) nodule from patient with TMNG Cold nodular tissues n 5 66.1^16.2* 76.9^18.3 (no. 3 in Table 1); lanes 5, 6, 7 non-nodular (normal), ˆ † hypofunctioning (cold) nodule andˆ hyperfunctioning (hot) nodule from patient with TMNG (no. 10 in Table 1); lane 8 positive Statistical analysis was performed using the paired Student's t-test: ˆ *P , 0:05: control. www.eje.org

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`cold' nodules. The resulting phenotype is represented by a large spectrum of clinical manifestations of thyrotoxicosis (14). The pathogenesis of the hot nodules of TMNG does not seem to differ from that of toxic adenomas, being related to the constitutive activation of the cAMP pathway due to mutations in the TSH receptor gene. Although, as in toxic adenomas, unequal prevalence in different studies and the large heterogeneity in the phenotypes challenge this theory (15±17). However, the role of cAMP in inducing at least an increase in iodide uptake, partially due to the stimulation of NIS gene and protein expression, is suggested by several in vitro studies in both rat and human cells (18±21). In contrast, molecular alterations that lead to the devel- opment of cold nodules remain unclear. Except for the reduced/lost ability to concentrate radioiodine, cold nodules, even in a given patient, may differ for clonality, growth rate, TSH dependence and expression of tissue-speci®c markers (22). In TMNG, nodules showing different behaviours regarding iodide trapping are present in a given patient. For this reason, this disease represents a unique and enviable in vivo model to study the molecular mechanisms underlying this process, limiting the interferences of both genetic and environmental factors. Figure 4 (A) Western blot analysis under reduced conditions of Alterations in the expression of the NIS gene have protein extracts from nodular and non-nodular tissues from four already been demonstrated in different thyroid pathol- patients with MNG, using anti-NIS (upper panel), anti-pendrin ogies, with increased levels in Graves' disease and toxic (middle panel) and anti-beta actin (lower panel) antibodies. A representative of three separate experiments is shown. adenomas, and decreased or undetectable levels in N normal thyroid tissue; C hypofunctioning (cold) nodule. (B) benign and malignant cold nodules (reviewed in 10, Levelsˆ of NIS and pendrin proteinˆ in hypofunctioning nodules of 11, 23), including metastases of differentiated thyroid patients with MNG. Values are calculated as in Fig. 2. Statistical carcinomas (24), with a high variability among analysis was performed as described in Materials and methods. different samples (25, 26). Since an important auto- regulatory effect of thyroid function is played by the intra-thyroidal iodide after its organi®cation (27), Correlation of NIS and pendrin expression in apical transport to the follicular lumen should also be each patient with TMNG considered. The recent characterization of the genetic alteration causing Pendred syndrome, a hereditary By analyzing the data from each patient, the expression disease characterized by neurosensorial deafness and of the two iodide transporters did not correlate in either goiter, allowed for the identi®cation of pendrin, a hot or cold nodules (data not shown). Thus, as shown chloride/iodide transporter mainly expressed in the in Fig. 2, in the hot nodules with the maximum thyroid and involved in iodide transport at the apical increase of NIS expression (more than two times over membrane of the thyrocytes (4, 5). Modi®cations in the the control, nos 4, 7 and 9), pendrin expression was PDS gene transcript have been demonstrated in some not increased to the same extent. Similarly, the cold thyroid diseases, with the most relevant reductions nodules with the lowest levels of NIS (nos 3 and 9), detected in thyroid carcinomas (8). showed increased pendrin expression. Our data show that, when compared with the non- nodular tissues, the highest levels of NIS expression were detected in the hot nodules, con®rming our Discussion previous data obtained in isolated toxic adenomas, both In iodine-de®cient areas, TMNG has a high prevalence at the protein level with immunohistochemical analysis and is responsible for a high percentage of cases of (6) and at the mRNA level with RT-PCR (26, 28). As thyrotoxicosis. Histologically, they are characterized by expected, inter-individual variability was also detected the presence of multiple hyperplastic nodules and well among the samples, as well as an absence of relation- encapsulated adenomas, with at least one nodule ship with the degree of thyrotoxicosis and other clinical characterized by TSH-independent growth, iodide parameters. In cold nodules, NIS expression was similar uptake and function, coexisting with one or more to the non-nodular tissue; this ®nding may result from

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Downloaded from Bioscientifica.com at 10/02/2021 04:05:42AM via free access 596 D Russo and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2001) 145 decreased NIS expression in normal tissue, owing to grants from the Associazione Italiana per la Ricerca sul TSH suppression. Cancro (SF), from MURST (SF and DR) and from Inter-individual heterogeneity is also present in the Electricite de France (MS). F Arturi is a recipient of expression of pendrin in TMNG tissues. However, it was Dottorato di Ricerca in `Basi molecolari dell'azione slightly increased in hot nodules (,1.5-fold) and, ormonale' at University of Catania. surprisingly, to the same extent in cold nodules, as compared with normal non-nodular tissue. Again, this ®nding may be related to decreased pendrin expression in normal tissue, due to TSH suppression. Indeed, these References data suggest a complex regulatory mechanism of 1 Carrasco N. Iodide transport in the thyroid gland. 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