help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-0805
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bernier-Valentin, F.
Right arrow Articles by Rousset, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bernier-Valentin, F.
Right arrow Articles by Rousset, B.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Endocrinology Vol. 147, No. 4 2035-2042
Copyright © 2006 by The Endocrine Society

Three-Dimensional Organization of Thyroid Cells into Follicle Structures Is a Pivotal Factor in the Control of Sodium/Iodide Symporter Expression

Françoise Bernier-Valentin, Séverine Trouttet-Masson, Rachida Rabilloud, Samia Selmi-Ruby and Bernard Rousset

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 369, F-69372 Lyon, France; Faculté de Médecine Laennec, F-69372 Lyon, France; and Université Lyon, F-69200 Lyon, France

Address all correspondence and requests for reprints to: Dr. Bernard Rousset, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 369, Faculté de Médecine Laennec, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France. E-mail: u369{at}sante.univ-lyon1.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of sodium/iodide symporter (NIS) by thyroid epithelial cells is primarily regulated by TSH, which acts at the level of NIS gene transcription. Knowledge of the mechanisms governing NIS expression mainly comes from studies of rat thyroid-derived cell lines forming cell monolayers. In this study we investigated the impact of the three-dimensional organization of thyroid cells into follicles on the regulation of NIS expression. We used porcine thyrocytes in primary culture that, depending on cell density and the moment TSH is added, either predominantly form a cell monolayer (CM) or reconstitute thyroid follicles (RTF). NIS expression analyzed at transcript and protein levels was remarkably high in RTF compared with CM. Cells forming RTF were NIS positive, whereas in CM, NIS was only detected in the limited number of cells forming follicle-like structures. When thyrocytes were cultured at increasing cell density to obtain a gradual shift from CM to RTF, the progressive increase in the proportion of cells enrolled in RTF was accompanied by a parallel increase in NIS expression. Other TSH-regulated genes, thyroperoxidase, Na+,K+-adenosine triphosphatase {alpha}-subunit, and thyroglobulin, were expressed at similar levels whatever the organization of thyrocytes in culture. The transcription factor, Pax-8, was equally expressed in NIS-negative CM and NIS-positive RTF. We show that TSH highly activates NIS expression only when thyrocytes have undergone histiotypic morphogenesis. This finding suggests that TSH activation of NIS gene transcription might involve, in addition to Pax-8, a regulatory factor(s) whose synthesis and/or activity are triggered by cell-cell interaction(s) occurring in the course of folliculogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IODIDE UPTAKE, the initial step of thyroid hormone biosynthesis, is an active transport process mediated by the sodium/iodide symporter (NIS). NIS belongs to the solute carrier family, in which it is designated SLC5A5 (1). NIS exerts its function of iodide transporter at the basolateral plasma membrane of thyrocytes organized in morphofunctional units, the thyroid follicles. Because iodide supply is the first limiting step in the hormone biosynthesis pathway, variations in the level of expression or the activity of NIS directly influence thyroid iodine economy. Expression of NIS by thyroid epithelial cells is regulated by TSH. This has been documented for rat NIS, both in vivo (2, 3) and on the rat thyroid-derived FRTL-5 cell line (2, 4, 5), and for human NIS using human thyrocytes in primary culture (6, 7, 8). Recent studies of TSH receptor-null mice (9, 10) also show that the expression of NIS in the thyroid is tightly dependent on TSH. TSH exerts its regulatory action through a cAMP-mediated activation of NIS gene transcription (11) The regulatory element responsible for the TSH-regulated transcriptional activation of NIS was first identified on the rat NIS gene (11); it is an enhancer (located 2–3 kbp upstream the transcription start site) named NIS upstream enhancer (NUE). Similar TSH-responsive NUE have now been described in human and mouse NIS genes (12, 13). The activity of NUE depends on its ability to bind the thyroid-specific transcription factor, Pax-8 (at two distinct sites), and cAMP response element (CRE)-like sequence binding proteins. TSH-induced activation of NIS transcription requires functional interaction between Pax-8 and other nuclear factors. The group of R. Di Lauro (14) recently reported that the CRE-like sequence of NUE is capable of interacting with numerous members of the activating protein-1 (AP-1) and CRE-binding protein family of transcription factors, indicating that this response element could conduct transcriptional regulation from diverse classes of cell signals and signaling pathways.

In this article we report that a signal originating from selective interactions between thyrocytes leads to a modulation of the transcriptional regulation of NIS by TSH. Using porcine thyrocytes in primary culture that have the ability to form a cell monolayer or to reconstitute thyroid follicles, we show that TSH highly activates NIS expression only when thyrocytes have undergone folliculogenesis. Analyses of NIS expression in the porcine species were made possible by our preceding work of cloning of the porcine NIS gene (15) from which we obtain information to generate antibodies recognizing porcine NIS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Thyroid cell culture
Thyroid glands of adult pigs were obtained from the local slaughterhouse and processed within 1 h of death. Freshly dispersed thyrocytes, prepared as previously described (16), were cultured in Ham’s F-12 medium (Seromed Biochrom KG, Berlin, Germany) supplemented with penicillin (200 U/ml), streptomycin (200 µg/ml), amphotericin B (0.5 µg/ml), and 10% calf serum (Invitrogen Life Technologies, Inc., Cergy-Pontoise, France) at 37 C under an air/CO2 (95%/5%) atmosphere. Cells were seeded at a density of 0.5 x 106 cells/cm2 in the absence or presence of TSH (1 mU/ml) (Sigma-Aldrich Corp., St Louis, MO). When present, TSH was added from the onset of culture to obtain reconstituted thyroid follicles (RTF) (16, 17) or after 1 d of culture; in this condition, thyrocytes formed a cell monolayer (CM). Thyrocytes cultured in the absence of TSH also formed a cell monolayer [CM(–)].

Production of antiporcine NIS antibodies
A peptide corresponding to a hydrophilic sequence located in the C-terminal domain (amino acids 621–635) of porcine NIS was generated by solid phase synthesis (Neosystem, Strasbourg, France). This peptide is common to the two main forms of porcine NIS generated by alternative splicing (15) and representing more than 95% of total NIS. The purified peptide, conjugated to keyhole limpet hemocyanin using glutaraldehyde, was used to immunize rabbits according to standard procedures. The antipeptide antibody titer was tested by ELISA, and immune serum pAb 72 was selected for immunological detection of porcine NIS by Western blot and immunofluorescence labeling. Antiporcine NIS antibodies were purified on the immobilized peptide using Affigel 15 (Bio-Rad Laboratories, Inc., Hercules, CA) and were stored at –20 C.

Western blot analysis
Thyroid cells in primary culture were washed three times in Earle’s medium (pH 7.0), collected by scraping in PBS supplemented with protease inhibitors (aprotinin, pepstatin, and leupeptin, each at a concentration of 1 µg/ml), and lysed by freezing and thawing in liquid nitrogen. Homogenates were centrifuged at 100,000 x g for 30 min at 4 C. The 100,000 x g pellet (crude membrane protein) was suspended in PBS supplemented with protease inhibitors, assayed for protein by the Lowry method after solubilization in 0.1% deoxycholate as previously reported (4) and frozen in aliquot samples at –80 C. Membrane proteins were fractionated by SDS-PAGE and analyzed by Western blot as previously described (4, 18) using affinity-purified anti-porcine NIS antibodies or antiporcine thyroperoxidase (TPO) antibodies (provided by J. Pommier, Institut National de la Santé et de la Recherche Médicale, Unité 96, Le Kremlin-Bicêtre, France). Immune complexes were detected with peroxidase-labeled secondary antibodies and a chemiluminescent reaction (ECL kit, Amersham Biosciences, Little Chalfont, UK). Images, digitized with a 300-ppi resolution on an 8-bit gray scale, were quantified with Image J software (National Institutes of Health, Bethesda, MD).

Iodide Uptake measurements
Thyroid cells cultured in petri dishes (6 cm in diameter) were washed twice in Earle’s medium (pH 6.8) supplemented with 1 mM methimazole and incubated in 1 ml of the same medium containing 0.5–1.0 µCi carrier-free Na125I (PerkinElmer Life Sciences, Courtaboeuf, France) and 10–6 M sodium iodide for 60 min at 37 C. Incubations were performed in the absence or presence of sodium perchlorate (0.1 mM) to determine the active iodide transport. At the end of the incubation period, cells were quickly washed with ice-cold Earle’s medium. The procedure was performed within 40 sec. Cells were scraped from dishes in PBS, and the suspension was counted in a {gamma}-counter (Packard Instruments Co., Groningen, The Netherlands). Incubations were made in triplicate. Iodide uptake was expressed as picomoles of iodide taken up per 106 cells. Cell number was determined from DNA fluorescence assay using Hoechst 33258 reagent (Molecular Probes, Eugene, OR) and calf thymus DNA as standard and considering that a cell contains 10 pg DNA.

Northern blot analysis
Total RNA was extracted by the acidic phenol-guanidinium isothiocyanate method. Samples of 25 µg total RNA were separated by electrophoresis in 1% agarose gel under denaturing conditions, transferred on Hybond+ nylon membrane, and hybridized with the following probes: a 0.8-kb porcine NIS cDNA generated by RT-PCR (15), a 1.6-kb cDNA fragment corresponding to the N-terminal half of the {alpha}1-subunit of rat Na+,K+-adenosine triphosphatase (ATPase) (19), a 0.6-kb cDNA corresponding to the N-terminal part of human thyroglobulin, extracted from the M1 plasmid (20) by PstI digestion (provided by Y. Malthierry, Angers, France), or a 0.7-kb cDNA fragment corresponding to the C-terminal portion of porcine TPO prepared from the PJ plasmid (21) using EcoRI and SacII (provided by B. Rapoport, Department of Medicine, Veterans Administration Medical Center, San Francisco, CA). Probes were labeled with [{alpha}-32P]deoxy-CTP (PerkinElmer Life Sciences) using the random oligonucleotide primed synthesis kit from Roche (Mannheim, Germany). After hybridization and washings, membranes were exposed to ß-radiation-sensitive screen. Signals were obtained using a PhosphorImager (Molecular Dynamics, Orsay, France; Centre Commun d’Imagerie Laennec, Lyon, France) and were quantified using ImageQuant software.

Indirect immunofluorescence
Cryosections of porcine thyroid tissue or cultured thyrocytes attached to petri dishes, fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.05% Tween 20, were incubated with affinity-purified antiporcine NIS antibodies (4 µg/ml), anti-TPO antibodies (immune serum at a 1:200 dilution), or a rat monoclonal anti-ZO1 antibody (1:200; from Chemicon International, Inc., Temecula, CA) or anti-Pax8 anti-peptide antibodies (pAb 791) generated in rabbits against the peptide amino acids 291–309 of the human Pax-8 sequence. Immune complexes were detected with fluorescein isothiocyanate (FITC)-labeled antirabbit or antirat Ig. Nuclei were stained with Hoechst 33342 reagent (Molecular Probes). Phase contrast and fluorescent images were obtained on an Axiovert 35M inverted microscope or an Axiophot microscope (Zeiss, Oberkochen, Germany), using a cooled CCD camera (Lhesa Electronique, Cergy-Pontoise, France) and Adobe image acquisition software (Adobe Systems, San Jose, CA).

Confocal microscopy
A laser scan confocal microscope LSCM 510 (Zeiss; Centre Commun de Quantimétrie, Domaine Rockefeller, Lyon, France) was used to analyze the cell polarity and three-dimensional organization of thyrocytes after labeling with anti-ZO1 antibodies and an FITC conjugate. An argon laser beam ({lambda} = 488 nm) was used for excitation of FITC, the emission filter had a bandpass of 510–563 nm. The top of cells cultured as CM or the top of follicular structures (RTF) was determined by prescanning, then the specimen was scanned from top to bottom to obtain optical sections of planes parallel to the bottom of the culture dish. Sequential images taken at 1-µm intervals in the z-axis were recorded and processed with the laser scan confocal microscope reconstruction program and Image J software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antibodies raised against the peptide (amino acids 621–635) located in the C-terminal domain of porcine NIS specifically identified 80- to 85-kDa molecular species in the 100,000 x g membrane fraction prepared from porcine thyroid tissue or porcine thyrocytes in culture. Like rat NIS (2, 4) or human NIS (18), porcine NIS gave rise to a 50- to 55-kDa species upon deglycosylation by N-glycosidase F (Fig. 1Go, A and B). The antiporcine NIS antibodies primarily labeled the basolateral plasma membrane of thyrocytes. This is illustrated in Fig. 1Go, E and F.


Figure 1
View larger version (118K):
[in this window]
[in a new window]
  		FIG. 1. Characterization of porcine NIS. A, Identification of porcine NIS by Western blot. Membrane protein fractions (50 µg) prepared from thyrocytes cultured for 4 d in the presence of TSH (lane 1), from porcine thyroid tissue (lane 2), or from porcine kidney (lane 3) were fractionated by SDS-PAGE, transferred onto an Immobilon-P membrane, and incubated with affinity-purified antibodies (0.4 µg/ml) directed against the peptide amino acids 621–635 of the porcine NIS sequence. B, Evidence that porcine NIS is a glycoprotein. Membrane protein samples (50 µg) prepared from thyrocytes cultured for 4 d with TSH were incubated with 0 (lanes 1 and 7), 3 (lane 2), 10 (lane 3), 30 (lane 4), 100 (lane 5), or 300 (lane 6) U/ml N-glycosidase F for 30 min at 37 C (lanes 1–6) or 4 C (lane 7) and analyzed by Western blot as described in A. The position of proteins of known molecular mass (expressed in kilodaltons) is shown on the right of each panel. C, Image of a paraffin section of porcine thyroid tissue. FL, Follicle lumen. D–F, Immunolocalization of TPO (D) and NIS (E and F) on porcine thyroid tissue cryosections. The anti-TPO antibodies labeled the apical pole of thyrocytes facing the follicle lumena (FL), whereas affinity-purified anti-porcine NIS antibodies (4 µg/ml) labeled the basal pole and the lateral domain of thyrocytes; this is clearly apparent on the enlarged field shown in E. Bar, 50 µm in C; 5 µm in D–F.

 
Regulation of porcine NIS expression by TSH
Changes in the expression of NIS by porcine thyrocytes in response to TSH were analyzed at transcript, protein, and activity (iodide uptake) levels over a 6-d period of culture (Fig. 2Go). Freshly isolated thyrocytes contained the 3.5- and 3.0-kb NIS transcript (15) and the 80- to 85-kDa NIS protein and exhibited low iodide uptake activity. During the first 24–36 h of culture, there was a decrease in NIS expression whatever the culture condition (presence or absence of TSH). On d 2 and thereafter, thyrocytes cultured in the absence of TSH had lost their capacity to trap iodide, and NIS transcript and protein were not detected (data not shown). By contrast, NIS expression was restored and became elevated (from d 3) in thyrocytes cultured with TSH (Fig. 2Go). Unexpectedly, thyrocytes cultured in the presence of TSH from the onset of culture, which formed RTF, exhibited very high and stable levels of NIS transcript and protein compared with thyrocytes cultured with TSH from d 1 and forming CM (Fig. 2Go). There was a 5- to 8-fold difference in both transcript and protein contents. This difference in NIS expression correlated with a 5-fold difference in the iodide uptake activity of RTF compared with CM. Thyrocytes cultured as either RTF or as CM were subjected to NIS immunolabeling (Fig. 3Go). Under RTF culture conditions, the vast majority of cells appeared involved in three-dimensional follicle structures, and these cells were NIS positive (Fig. 3CGo). As in intact thyroid tissue, anti-NIS antibodies preferentially labeled the basolateral plasma membrane of thyrocytes participating in follicle structures (Fig. 3DGo). In CM, the large majority of cells formed a monolayer, but there were foci of cell aggregates or follicle-like structures. It was clearly apparent that NIS was mainly expressed by the limited number of cells forming aggregates or follicle-like structures. A low labeling was detectable, however, over thyrocytes spread on the dish. These observations suggested that the level of expression of NIS by porcine thyrocytes in culture could be linked to their reorganization in follicle structures. Because thyrocytes involved in the formation of three-dimensional structures are polarized, we examined whether the difference in NIS expression between RTF and CM could simply be due to the lack of cell polarity in CM. The data in Fig. 4Go demonstrate that cells forming CM or RTF had established tight junctions visualized by ZO1 immunolabeling, and that in both cases, thyrocytes had reacquired a polarized epithelial phenotype.


Figure 2
View larger version (46K):
[in this window]
[in a new window]
  		FIG. 2. NIS expression by porcine thyrocytes in primary culture: regulatory actions of TSH. Freshly isolated porcine thyrocytes, seeded at a density of 0.5 x 106 cells/cm2, were cultured for 1–6 d in the absence of TSH or in the presence of 1 mU/ml TSH added from the onset of culture (RTF) or after 1 d of culture (CM). A, Changes in iodide uptake (IU) activity of porcine thyrocytes over the 6-d period. Symbols and vertical bars represent the mean and SEM of triplicate determinations. {circ}, Thyrocytes cultured without TSH; bullet, thyrocytes cultured as RTF; {blacktriangleup}, thyrocytes cultured as CM. Only part of the data (d 2 and 6) corresponding to thyrocytes cultured in the absence of TSH are shown. B and D, Time-dependent variations in the NIS transcript content of porcine thyrocytes cultured as RTF or CM. The Northern blot analysis comprised two successive hybridization steps with a 32P-labeled NIS cDNA probe and then with a 32P-labeled ß-actin probe. B, Porcine NIS transcripts of 3.0 and 3.5 kb are identified by arrows, and the ß-actin transcript is indicated by arrowheads. NIS mRNA signals were quantified and normalized to the ß-actin transcript signal; values are presented in D. Data obtained from thyrocytes cultured without TSH on d 2 and 6 are also shown. Symbols are the same as in A. C and E, Time-dependent variations of the NIS protein content of porcine thyrocytes cultured as RTF or CM. Membrane protein (50 µg) was analyzed by Western blot as described in Fig. 1Go. The intensity of the 80- to 85-kDa band was quantified, and values are reported in E. Data obtained from thyrocytes cultured without TSH on d 2 and 6 are also shown.

 

Figure 3
View larger version (59K):
[in this window]
[in a new window]
  		FIG. 3. Immunodetection of NIS expressed by porcine thyrocytes in primary culture. Porcine thyrocytes cultured for 4 d in the absence (A) or presence of TSH from d 1 of culture (B) or from the onset of culture (C and D) were processed for NIS detection by indirect immunofluorescence using affinity-purified antiporcine NIS antibodies (4 µg/ml) and an FITC-labeled secondary antibody. Nuclei were labeled using Hoechst 33342 reagent. Immunofluorescence images (A1, B1, C1, and D1) and Hoechst fluorescence images (A2, B2, and C2) or a phase contrast image (D2) of the same microscope fields are shown. Bars, 25 µm in A–C; 50 µm in D. The organization of thyrocytes in the culture dish, i.e. cell monolayer or follicle structure, can easily be deduced from the distribution of labeled nuclei.

 

Figure 4
View larger version (94K):
[in this window]
[in a new window]
  		FIG. 4. Confocal microscopic analysis of tight junction formation in porcine thyrocytes in primary culture. Thyrocytes cultured as CM or RTF for 5 d were processed for ZO1 detection by indirect immunofluorescence using a monoclonal anti-ZO1 antibody and an FITC-labeled secondary antibody. Fluorescence images of CM (A) and RTF (B) were reconstructed from serial optical sections (parallel to the surface of the dish) collected in the z orientation (from the top to the bottom of the dish). In both culture conditions, thyrocytes exhibited continuous tight junction belts identified by the tight junction-associated ZO1. The white horizontal lines in A and B define the locations of the transverse optical sections presented with a 2-fold enlargement in C and D, respectively. C, Tight junctions of thyrocytes cultured as CM appear as vertical segments rather regularly spaced at some distance from the bottom of the dish; this is in keeping with a near-apical location of tight junctions on the lateral plasma membrane of thyrocytes. D, Tight junctions serve to visualize the three-dimensional structure of RTF. The space located between the upper and the lower cell layers corresponds to the internal luminal compartment of the reconstituted thyroid follicle. The white arrows indicate the bottom of the culture dish.

 
Relationship between NIS expression and reconstitution of thyroid follicles
The impact of thyroid cell organization on TSH-induced NIS expression was directly analyzed by culturing thyrocytes at various densities. As shown in Fig. 5Go, the proportion of thyrocytes participating in three-dimensional follicle structures progressively increased with the cell seeding density. At a cell density of 0.125 x 106 cells/cm2, thyrocytes exclusively formed a cell monolayer, and at a density higher than 0.5 x 106 cells/cm2, the quasi-totality of thyrocytes turned into follicles. The results of NIS expression analyses under these various culture conditions are presented in Fig. 6Go. At the lowest cell density (0.125 and 0.166 x 106 cells/cm2), NIS mRNAs and protein were barely detectable. The increase in cell density leading to an increase in follicle formation was accompanied by a gradual and parallel increase in NIS transcript and NIS protein cell contents (Fig. 6Go, B, C, and E). Noteworthy, iodide uptake activity of thyrocytes (Fig. 6AGo) did not follow exactly the same variations. Thyrocytes, exclusively as CM, with an NIS content below the detection limit, exhibited a definite capacity to trap iodide, and the maximum iodide uptake activity was reached at a cell density that led to only partial follicle formation.


Figure 5
View larger version (99K):
[in this window]
[in a new window]
  		FIG. 5. Reconstitution of thyroid follicles as a function of thyrocyte seeding density. Freshly isolated porcine thyrocytes seeded at increasing density: [0.125 (A), 0.166 (B), 0.25 (C), 0.33 (D), 0.5 (E), and 0.75 (F) x 106 cells/cm2] were cultured with 1 mU/ml TSH for 4 d. A–F, Representative phase contrast images of thyroid cell organization.

 

Figure 6
View larger version (38K):
[in this window]
[in a new window]
  		FIG. 6. Changes in the level of expression of NIS as a function of thyrocyte seeding density. Freshly isolated thyrocytes were seeded at increasing cell density (from 0.125–0.75 x 106 cells/cm2) and cultured in the presence of TSH (1 mU/ml) for 4 d as described in Fig. 5Go. A, Iodide uptake (IU) activity of thyrocytes cultured at various densities. Symbols and vertical bars represent the mean and SEM of triplicate determinations. B, Northern blot analysis of the NIS transcript content of thyrocytes cultured at increasing density [0.125 (lane 1), 0.166 (lane 2), 0.25 (lane 3), 0.33 (lane 4), 0.5 (lane 5), and 0.75 (F) x 106 cells/cm2]. Lane 7 corresponds to thyrocytes (0.5 x 106 cells/cm2) cultured in the absence of TSH. Ethidium bromide-stained 28S RNA is shown as a quantitative reference of RNA loading. C and D, Western blot analyses of NIS and TPO contents of thyrocytes cultured at various densities as described in B. E, Quantitative analyses of the changes in NIS transcript (bullet), NIS protein ({blacktriangleup}), and TPO ({blacksquare}) as a function of the cell seeding density. Values were obtained by quantification of the signals shown in B–D. {square} and {circ}, Values obtained from cells cultured in the absence of TSH (lanes 7 of B–D). Data from a representative experiment of three are shown.

 
Influence of three-dimensional organization of thyrocytes on the expression of other TSH-regulated genes
Thyrocytes cultured at various cell densities were analyzed for their TPO content. As shown in Fig. 6Go, D and E, TSH activated TPO expression to about the same extent whatever the organization of thyrocytes. This is also demonstrated by the immunolabeling experiments shown in Fig. 7Go. In agreement with the Western blot data in Fig. 6Go, NIS labeling was intense over follicle structures and was low or barely detectable in monolayer cells, whereas TPO labeling had the same apparent intensity in monolayer cells and cells constituting follicle structures. This study was extended to other TSH-regulated genes (Fig. 8Go). Thyroglobulin and Na+,K+-ATPase {alpha}-subunit transcripts were similarly activated by TSH in CM and RTF.


Figure 7
View larger version (77K):
[in this window]
[in a new window]
  		FIG. 7. Relationship between NIS expression and the three-dimensional organization of thyrocytes into follicle structures. Porcine thyrocytes seeded at different cell densities (0.125, 0.166, and 0.33 x 106 cells/cm2) and cultured in the presence of TSH (1 mU/ml) from the onset were processed for NIS or TPO detection by indirect immunofluorescence using the same antibodies as in the Western blot and an FITC-labeled secondary antibody. For each experimental condition, the upper image corresponds to immunolabeling, and the lower image corresponds to the labeling of nuclei (Hoechst 33342 fluorescence) of the same microscope field.

 

Figure 8
View larger version (97K):
[in this window]
[in a new window]
  		FIG. 8. Influence of thyroid cell organization on the expression of different TSH-regulated genes. Porcine thyrocytes (0.5 x 106 cells/cm2) were cultured in the absence [CM(–)] or presence of TSH as CM or RTF for 4 d and analyzed for NIS, thyroglobulin, TPO, and Na+,K+-ATPase {alpha}-subunit transcript contents by Northern blot, using probes described in Materials and Methods. Ethidium bromide staining of ribosomal RNA or hybridization with a ß-actin probe were used as a control of RNA loading.

 
Differential TSH-induced activation of NIS expression in cell monolayers and thyroid follicles is not related to a difference in Pax-8 expression or cell distribution
The transcription factor Pax-8 is a mandatory component for the transactivation of NIS gene transcription in thyroid epithelial cells (11). As previously reported in dog (22) and rat (23) thyrocytes, we show that the expression of Pax-8 by porcine thyrocytes is dependent on TSH stimulation; thyrocytes cultured in the absence of TSH [CM(–)] were Pax-8 negative (Fig. 9Go). In the presence of TSH, added either from the onset of culture (RTF) or on d 1 (CM), thyrocytes similarly expressed Pax-8, which was essentially located in the nucleus.


Figure 9
View larger version (89K):
[in this window]
[in a new window]
  		FIG. 9. Expression of the transcription factor Pax-8 is activated by TSH regardless of thyroid cell organization. Porcine thyrocytes cultured in the absence [CM(–)] or presence of TSH as CM or RTF for 4 d were processed for Pax-8 detection by indirect immunofluorescence using anti-Pax8 antipeptide antibodies and an FITC-labeled secondary antibody. A phase contrast image (on the left) and an immunofluorescence image (on the right) of the same microscope field are shown. When expressed, Pax-8 exhibited a predominant nuclear localization.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Taking advantage of the rather unique property of isolated porcine thyrocytes to reproduce in vitro the thyroid differentiation program leading to folliculogenesis, we have evidenced a new complexity in the control of NIS expression. We show that TSH highly activates NIS expression only when thyrocytes are committed to form three-dimensional follicle structures. TSH-stimulated thyrocytes outside these structures exhibit minimal expression of NIS. The mechanism that links thyroid folliculogenesis to NIS expression should operate at the level of transcription. Indeed, folliculogenesis-related changes in NIS transcript levels kinetically and quantitatively preceded changes in NIS protein. The modulation of NIS expression associated with follicle morphogenesis appears selective, because the level of expression of other TSH-regulated genes was not affected by the state of organization of thyrocytes. Two membrane proteins with distinct subcellular location, i.e. TPO, which is delivered at the apical plasma membrane, and Na+,K+-ATPase {alpha}-subunit, which is targeted at the basolateral plasma membrane and participates in the constitution of Na+,K+-ATPase {alpha}-ß active complex, were expressed at similar levels in CM and RTF. These findings suggest that the activation of NIS gene transcription by TSH might involve a regulatory factor(s) whose synthesis and/or activity is triggered by cell-cell interactions occurring selectively in the course of folliculogenesis.

Because thyrocytes cultured in the form of CM or RTF both exhibited the polarized epithelial phenotype, the establishment of cell polarity should not represent an important determinant of the TSH-induced enhancement of NIS expression. Previous studies of porcine thyrocytes have revealed that follicle morphogenesis first requires the formation of three-dimensional cell aggregates within which cell polarization occurs and a luminal compartment emerges (24, 25, 26). Thus, one can reasonably postulate that the process that triggers the TSH-induced enhancement of NIS expression could precede the establishment of cell polarity; it could take place during the initial phase of folliculogenesis, i.e. the step of cell-cell adhesion leading to the formation of aggregates. The initiation of cell-cell contacts is known to be regulated by cell adhesion molecules. The adhesion between isolated porcine thyrocytes that occurs during the first 10–20 h after cell seeding is dependent upon the plasma membrane protein, E-cadherin (26). It was reported that the addition of a monoclonal antibody directed against the extracellular domain of E-cadherin to the culture medium of porcine thyrocytes prevented cell aggregation (26). Because TSH activates the expression of E-cadherin (27) and stabilizes its assembly and retention at the surface of porcine thyrocytes (28), the TSH-induced enhancement of NIS expression might originate from E-cadherin-mediated aggregation of thyrocytes. E-cadherin has the ability to alter gene expression indirectly by interacting with ß-catenin and thus interfering with the regulatory action of ß-catenin-T cell factor/lymphoid enhancer factor complex on the transcription of target genes, including components of the AP-1 transcription complex (29). A functional interaction between ß-catenin and members of the T cell factor/lymphoid enhancer factor family has been found in thyrocytes (30). The possible implication of E-cadherin in the control of NIS expression by porcine thyrocytes will not be easy to test using approaches based on the expression of exogenous genes or interfering RNA. Indeed, the transfection efficiency of porcine thyrocytes in primary culture is low and, in our hands, always leads to a loss of the ability of thyrocytes to reconstitute follicles.

The iodide uptake activity of porcine thyrocytes in culture was globally related to the NIS protein cell content with a few notable exceptions however. During the first 24 h of culture, without or with TSH, there was a marked decrease in NIS cell content, but the capacity of TSH-stimulated cells to concentrate iodide increased. This observation suggests that TSH could stimulate the activity of residual NIS through either posttranslational modifications (31) or a change in the concentration or activity of proteins regulating NIS functioning (4). A comparable dissociation between NIS expression level and iodide uptake activity in response to TSH was observed in long-term cultured human thyrocytes (7). The decline of NIS expression early in culture of porcine thyrocytes was not restricted to NIS; similar changes were repeatedly observed for most of the genes examined, including TPO and Pax-8. This general shut off of gene expression probably reflects the adaptation of cells to culture conditions after the proteolytic treatment used for cell isolation. A dissociation between NIS protein level and iodide uptake activity was also observed when thyrocytes were cultured at various cell densities. First, at the lowest cell seeding density, NIS transcripts and NIS protein were not detected, but thyrocytes (in the form of a monolayer) were capable of concentrating iodide; again, this observation suggests that TSH might exert control over NIS activity. Second, at the cell density that yielded about half-maximum NIS expression, the iodide uptake activity of thyrocytes was already maximum. This might indicate that only part of NIS is functional when expressed at high levels. NIS molecules may remain in intracellular compartments instead of being targeted to the plasma membrane.

In conclusion, by analyzing NIS expression in the porcine thyroid cell system, we confirm previous data obtained from rat thyroid cell lines and human thyrocytes in primary culture, and we report a new and probably physiologically relevant mechanism of control of NIS expression. The link between the expression of NIS and the morphogenetic differentiation program could represent a functional adaptation for adequate thyroid iodide supply. Thyrocytes would synthesize NIS and therefore concentrate iodide at a high rate only when they form the closed compartment, the follicle lumen, in which iodide can be conveyed and converted into hormonal iodine. In contrast, a lack of follicle organization or a disruption of follicle structures could lead to a slow down or a shut off of NIS expression; this might contribute to the down-regulation of NIS expression observed in human tumors (18, 32).


   Acknowledgments
 
We thank Catherine Limoge for her assistance with preparation of the manuscript.


   Footnotes
 
First Published Online December 8, 2005

Abbreviations: AP-1, Activating protein-1; ATPase, Na+,K+-adenosine triphosphatase; CM, cell monolayer; CM(–), cell monolayer formed in the absence of TSH; CRE, cAMP response element; FITC, fluorescein isothiocyanate; NIS, sodium/iodide symporter; NUE, NIS upstream enhancer; RTF, reconstituted thyroid follicles; TPO, thyroperoxidase.

Received June 29, 2005.

Accepted for publication November 29, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dohan O, de la Vieja A, Paroder V, Riedel C, Artani M, Reed M, Ginter CS, Carrasco N 2003 The sodium/iodide symporter (NIS): characterization, regulation, and medical significance. Endocr Rev 24:48–77[Abstract/Free Full Text]
  2. Levy O, Dai G, Riedel C, Ginter CS, Paul EM, Lebowitz AN, Carrasco N 1997 Characterization of the thyroid Na+/I– symporter with an anti-COOH terminus antibody. Proc Natl Acad Sci USA 94:5568–5573[Abstract/Free Full Text]
  3. Uttersprot N, Pelgrims N, Carrasco N, Gervy C, Maenhaut C, Dumont JE, Miot F 1997 Moderate doses of iodide in vivo inhibit cell proliferation and the expression of thyroperoxidase and Na+/I– symporter mRNA in dog thyroid. Mol Cell Endocrinol 131:195–203[CrossRef][Medline]
  4. Paire A, Bernier-Valentin F, Selmi-Ruby S, Rousset B 1997 Characterization of the rat thyroid iodide transporter using anti-peptide antibodies. J Biol Chem 272:18245–18249[Abstract/Free Full Text]
  5. Kogai T, Endo T, Saito T, Miyazaki A, Kawaguchi A, Onaya T 1997 Regulation by thyroid-stimulating hormone of sodium/iodide gene expression and protein levels in FRTL-5 cells. Endocrinology 138:2227–2232[Abstract/Free Full Text]
  6. Saiko T, Endo T, Kawaguchi A, Ikeda M, Nakazato M, Kogai T, Onaya T 1997 Increased expression of the Na+/I– symporter in cultured human thyroid cells exposed to thyrotropin and in Graves’ thyroid tissues. J Clin Endocrinol Metab 82:3331–3336[Abstract/Free Full Text]
  7. Kogai T, Curcio F, Hyman S, Cornford EM, Brent GA, Hershman JM 2000 Induction of follicle formation in long-term cultured normal human thyroid cells treated with thyrotropin stimulates iodide uptake but not sodium/iodide symporter messenger RNA and protein expression. J Endocrinol 167:125–135[Abstract]
  8. Porra V, Ferraro-Peyret C, Durand C, Selmi-Ruby S, Giroud H, Berger-Dutrieux N, Decaussin M, Peix JL, Bournaud C, Orgiazzi J, Borson-Chazot F, Dante R, Rousset B 2005 Silencing of the tumor suppressor gene SLC5A8 is associated with BRAF mutations in classical papillary thyroid carcinomas. J Clin Endocrinol Metab 90:3028–3035[Abstract/Free Full Text]
  9. Marians RC, Ng L, Blair HC, Unger P, Graves PN, Davies TF 2002 Defining thyrotropin-dependent and -independent steps of thyroid hormone synthesis by using thyrotropin receptor null-mice. Proc Natl Acad Sci USA 99:15776–15781[Abstract/Free Full Text]
  10. Postiglione MP, Parlato R, Rodriguez-Mallon A, Rosica A, Mithbaokar P, Maresca M, Marians RC, Davies FT, Zannini MS, De Felice M, Di Lauro R 2002 Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland. Proc Natl Acad Sci USA 99:15462–15467[Abstract/Free Full Text]
  11. Ohno M, Zannini M, Lévy O, Carrasco N, Di Lauro R 1999 The paired-domain transcription factor Pax-8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription. Mol Cell Biol 19:2051–2060[Abstract/Free Full Text]
  12. Taki K, Kogai T, Kanamoto Y, Hershman JM, Brent GA 2002 A thyroid-specific far-upstream enhancer in the human sodium/iodide symporter gene requires Pax-8 binding and cyclic adenosine 3',5'-monophosphate response element-like sequence binding proteins for full activity and is differentially regulated in normal and thyroid cancer cells. Mol Endocrinol 16:2266–2282[Abstract/Free Full Text]
  13. Lin X, Ryu KY, Jhiang SM 2004 Cloning of the 5'-flanking region of mouse sodium-iodide symporter and identification of a thyroid-specific and TSH-responsive enhancer. Thyroid 14:19–27[CrossRef][Medline]
  14. Chun JT, Dato D, d’Andréa B, Zannini M, Di Lauro R 2004 The CRE-like element inside the 5'-upstream region of the rat sodium/iodide symporter gene interacts with diverse classes of b-Zip molecules that regulate transcriptional activities through strong synergy with Pax-8. Mol Endocrinol 18:2817–2829[Abstract/Free Full Text]
  15. Selmi-Ruby S, Watrin C, Trouttet-Masson S, Bernier-Valentin F, Flachon V, Munari-Silem Y, Rousset B 2003 The porcine sodium/iodide symporter gene exhibits an uncommon expression pattern related to the use of alternative splice sites not present in the human or murine species. Endocrinology 144:1074–1085[Abstract/Free Full Text]
  16. Munari-Silem Y, Mesnil M, Selmi S, Bernier-Valentin F, Rabilloud R, Rousset B 1990 Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles. J Cell Physiol 145:414–427[CrossRef][Medline]
  17. Bernier-Valentin F, Kostrouch Z, Rabilloud R, Rousset B 1991 Analysis of the thyroglobulin internalization process using in vitro reconstituted thyroid follicles: evidence for a coated vesicle-dependent endocytic pathway. Endocrinology 129:2194–2201[Abstract]
  18. Trouttet-Masson S, Selmi-Ruby S, Bernier-Valentin F, Porra V, Berger-Dutrieux N, Decaussin M, Peix JL, Perrin A, Bournaud C, Orgiazzi J, Borson-Chazot F, Franc B, Rousset B 2004 Evidence for transcriptional and posttranscriptional alterations of the sodium/iodide symporter expression in hypofunctioning benign and malignant thyroid tumors. Am J Pathol 165:25–34[Abstract/Free Full Text]
  19. Paire A, Bernier-Valentin F, Rabilloud R, Watrin C, Selmi-Ruby S, Rousset B 1988 Expression of {alpha}- and ß-subunits and activity of Na+K+ATPase in pig thyroid cells in primary culture: modulation by thyrotropin and thyroid hormones. Mol Cell Endocrinol 146:93–101
  20. Malthiery Y, Lissitzky S 1987 Primary structure of human thyroglobulin deduced from the sequence of its 8448-base complementary DNA. Eur J Biochem 165:491–498[Medline]
  21. Magnusson RP, Gestautas J, Seto P, Taurog A, Rapoport B 1986 Isolation and characterization of a cDNA clone for porcine thyroid peroxidase. FEBS Lett 208:391–396[CrossRef][Medline]
  22. Van Renterghem P, Vassart G, Christophe D 1996 Pax 8 expression in primary cultured dog thyrocyte is increased by cyclic AMP. Biochem Biophys Acta 1307:97–103[Medline]
  23. Fabbro D, Pellizzari L, Mercuri F, Tell G, Damante G 1998 Pax-8 protein levels regulate thyroglobulin gene expression. J Mol Endocrinol 21:347–354[Abstract]
  24. Yap AS, Manley SW 1993 Contact inhibition of cell spreading: a mechanism for the maintenance of thyroid cell aggregation in vitro. Exp Cell Res 208:121–127[CrossRef][Medline]
  25. Yap AS, Stevenson BR, Armstrong JW, Keast JR, Manley SW 1994 Thyroid epithelial morphogenesis in vitro: a role for bumetanide-sensitive Cl– secretion during follicular lumen development. Exp Cell Res 213:319–326[CrossRef][Medline]
  26. Yap AS, Stevenson BR, Keast JR, Manley SW 1995 Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation. Endocrinology 136:4672–4680[Abstract]
  27. Brabant G, Hoang-Vu C, Behrends J, Cetin Y, Potter E, Dumont JE, Maenhaut C 1995 Regulation of the cell-cell adhesion protein, E-cadherin, in dog and human thyrocytes in vitro. Endocrinology 136:3113–3119[Abstract]
  28. Larsson F, Fagman H, Nilsson M 2004 TSH receptor signaling via cyclic AMP inhibits cell surface degradation and internalization of E-cadherin in pig thyroid epithelium. Cell Mol Life Sci 61:1834–1842[Medline]
  29. Mann B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, Bodmer WF, Moyer MP, Riecken EO, Buhr HJ, Hanski C 1999 Target genes of ß-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci USA 99:1603–1608
  30. Helmbrecht K, Kispert A, von Wasielewski R, Brabant G 2001 Identification of a Wnt/ß-catenin signaling pathway in human thyroid cells. Endocrinology 142:5261–5266[Abstract/Free Full Text]
  31. Riedel C, Lévy O, Carrasco N 2001 Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J Biol Chem 276:21458–21463[Abstract/Free Full Text]
  32. Lazar V, Bidart JM, Caillou B, Mahe C, Lacroix L, Filetti S, Schlumberger M 1999 Expression of the Na+/I– symporter gene in human thyroid tumors: a comparison study with other thyroid-specific genes. J Clin Endocrinol Metab 84:3228–3234[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
G. Szinnai, L. Lacroix, A. Carre, F. Guimiot, M. Talbot, J. Martinovic, A.-L. Delezoide, M. Vekemans, S. Michiels, B. Caillou, et al.
Sodium/Iodide Symporter (NIS) Gene Expression Is the Limiting Step for the Onset of Thyroid Function in the Human Fetus
J. Clin. Endocrinol. Metab., January 1, 2007; 92(1): 70 - 76.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
T Kogai, K Taki, and G A Brent
Enhancement of sodium/iodide symporter expression in thyroid and breast cancer.
Endocr. Relat. Cancer, September 1, 2006; 13(3): 797 - 826.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bernier-Valentin, F.
Right arrow Articles by Rousset, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bernier-Valentin, F.
Right arrow Articles by Rousset, B.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals