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Characterization and Semiquantitative Analyses of Pendrin Expressed in Normal and Tumoral Human Thyroid Tissues

INSERM, U-369, Faculté de Médecine Lyon RTH-Laennec (V.P., F.B.-V., S.T.-M., S.S.-R., B.R.); Unité Fonctionnelle de Biologie Cellulaire, Hôpital Edouard-Herriot (V.P., B.R.); and Lyon Thyroid Tumor Bank Organization (N.B.-D., J.-L.P., A.P.), 69372 Lyon, France

Address all correspondence and requests for reprints to: Prof. Bernard Rousset, INSERM, U-369, Faculté de Médecine Lyon-RTH Laennec, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France. E-mail: .

Abstract

The gene mutated in Pendred syndrome (PDS), the PDS gene, is expressed in the inner ear, kidney, and thyroid. It encodes a membrane protein named pendrin that is endowed with the function of anion transporter or exchanger. It has been postulated that in the thyroid pendrin could participate in the transport of iodide from the cell to the lumen of follicles. We generated antipeptide antibodies directed against the C- terminal sequence of human pendrin 1) to characterize the protein expressed in the human thyroid, and 2) to analyze its expression level in relation to the functional activity of thyroid tissue. In denaturing conditions, a single molecular species of 110–115 kDa was identified in human thyroid membrane fractions. After treatment of thyroid membranes with N-glycosidase F, pendrin had an apparent molecular mass of 85 kDa. Analyzed by ultracentrifugation on sucrose gradient in nondenaturing conditions, pendrin sedimented as a main 120- to 140-kDa component. Pendrin was assayed by semiquantitative Western blot in thyroid membrane fractions from 25 hyper- or hypofunctioning tumors and paired normal tissue samples. Pendrin was increased 2-fold in toxic adenomas, was not significantly altered in follicular adenoma, and was decreased, on the average, by 35% in papillary carcinomas compared with levels in paired normal tissue. The variations in the pendrin tissue content and PDS transcript levels, assayed by RT-PCR on duplicate samples of the same tumors, were similar. In conclusion, we show that pendrin expressed by the human thyroid gland is a mainly monomeric glycoprotein and that the level of expression of pendrin, although somewhat related, only moderately varied with the functional status of the thyroid tissue.

THE GENE RESPONSIBLE for Pendred syndrome (PDS), an autosomal recessive disorder characterized by congenital sensorineural deafness and goiter (1, 2), was identified 5 yr ago by positional cloning (3). As expected, it is expressed in thyroid gland and inner ear (3, 4) as well as in kidneys (3, 5), syncytiotrophoblasts (6), and Sertoli cells (7).

Pendrin, the protein encoded by this gene, contains 780 amino acids and is assumed to have 11 or 12 transmembrane domains (3, 8). By expressing the PDS gene in heterologous cell systems, it was demonstrated that pendrin was acting as a transporter of numerous anions, chloride, iodide, formate, and bicarbonate, but not sulfate, despite a close homology between this protein and many sulfate transporters (9, 10, 11). In the kidney cortex, pendrin functions as an anion transporter, mediating chloride/base exchange (5, 11). In the thyroid, pendrin is located at the apical plasma membrane of thyrocytes (8); it has been postulated that pendrin might be involved in the transport of iodide from the cell to the follicular lumen and that the loss of function of this anion transporter would be responsible for the partial organification defect observed in Pendred syndrome.

Recently, PDS gene expression has been studied in human hypofunctioning benign or malignant thyroid tumors (12); it was found that the PDS transcript level, analyzed by quantitative PCR, was decreased in carcinomas compared with normal thyroid tissue samples, but was not modified in hypofunctioning adenomas. The PDS transcript content of hyperfunctioning adenomas was also indistinguishable from that of a series of normal tissue (NT) samples.

In the present study we prepared antipeptide antibodies directed against the C-terminal sequence of human pendrin 1) to characterize the protein expressed by the human thyroid gland in terms of size in native and denatured states and glycosylation status, and 2) to examine the relationship between pendrin expression and the functional activity of thyroid tissue. To this purpose we compared the pendrin content of hyperfunctioning tumors [toxic adenomas (TA)], normal thyroid tissues, and hypofunctioning (benign or malignant) thyroid tumors. The study was carried out on 50 tissue samples consisting of 25 thyroid tumors (either hyper- or hypofunctioning) and the 25 paired samples of normal thyroid tissue. Our data demonstrate rather limited variations of the level of expression of pendrin with the functional status of thyroid tissue and show that papillary carcinomas (PC) continue to express the 110- to 115-kDa glycosylated pendrin.

Materials and Methods

Human thyroid tissues

Thyroid tissue samples were taken from the Lyon Thyroid Tumor Bank, which was established in 1998 as part of a collaborative clinical research project on differentiated thyroid cancer at the Lyon University Hospital Center. This study was approved by the supervision interdisciplinary committee of the Tumor Bank and performed in accordance with protocols previously approved by the local human studies committee. Specimens maintained in the bank consisted of pairs of samples: a fragment of thyroid tumor and a fragment of normal thyroid tissue selected at the time of extemporaneous examination of surgical pieces from patients undergoing partial or total thyroidectomy. Tissue samples weighing 50–200 mg were frozen in liquid nitrogen and stored at -80 C. Thyroid tissue samples selected after histological examination were classified according to WHO recommendations. Pathological and clinical annotations were anonymously associated with the samples maintained in the Tumor Bank.

Samples used in this study were TA (n = 3), follicular adenoma (FA; n = 4), and PC (n = 18) and paired NT (n = 25). Thyroid tissue samples were obtained from euthyroid subjects at the time of surgery, except TA from patients in whom serum TSH was below 0.05 mU/liter or undetectable, and radioiodine uptake in the nodule increased on thyroid scintigraphy. Thyroid scintigraphy was also performed in 2 of the 4 patients with FA and 11 of the 18 patients with PC; in all cases, the nodule appeared hypofunctioning. FA came from 2 males and 2 females (mean ± SD age, 43.7 ± 17.3 yr) and PC from 7 males and 11 females (mean age, 43.6 ± 15.9 yr). According to the TNM classification, 8 PC were classified as stage I, 6 as stage II, and 4 as stage III.

Preparation and test of antibodies

A peptide corresponding to the published sequence (3)(access number Swiss Protein Bank O43511) of the C-terminal region of pendrin (amino acid 766–780) was synthesized by a conventional solid phase procedure using an automated peptide synthesizer (Neosystem Laboratories, Strasbourg, France) and subsequently purified by high performance liquid chromatography. The identity of the peptide was checked by mass spectrometry and amino acid analysis. The resulting peptide was conjugated to keyhole limpet hemocyanin using glutaraldehyde as a coupling reagent. Antisera were raised in rabbits by multipoint injection of 200 µg conjugate emulsified in complete Freund’s adjuvant. After two boost injections at 3-wk intervals, animals were bled and sera were tested for the presence of antipeptide antibodies by ELISA, using the synthetic peptide coated on multiwell plates (Corning, Costar Corp., Cambridge, MA). Immune complexes were detected using an antirabbit Ig antibody conjugated to alkaline phosphatase and p-nitrophenyl phosphate as substrate (Sigma, St. Louis, MO). Alkaline phosphatase activity was assayed by absorbance measurements at 405 nm. One immune serum, pAb 827, was selected for subsequent studies. The IgG fraction from pAb 827 was purified by anion exchange chromatography on diethylaminoethyl-Sephacel (Pharmacia Biotech, Orsay, France).

Western blot analyses

Tissue samples frozen at -80 C were rapidly weighed, and homogenized using a Teflon-glass Potter homogenizer in ice-cold PBS (1 ml/100 mg tissue) supplemented with protease inhibitors (aprotinin, leupeptin, and pepstatin, each at a concentration of 1 µg/ml). Homogenates were centrifuged at 100,000 x g for 60 min at 4 C to obtain the crude thyroid membrane fractions. Protein was assayed by the Lowry method after solubilization in 0.1% sodium deoxycholate. Membrane proteins (from 3–180 µg proteins) were fractionated by electrophoresis on 8% acrylamide gel in the presence of SDS and electrotransferred onto Immobilon P membrane (Millipore Corp., Bedford, MA). After treatment with PBS supplemented with 5% nonfat dry milk and 0.2% Tween 20 (Sigma), membranes were incubated overnight at 4 C with 40 µg/ml IgG from pAb 827. After three washings in PBS-0.2% Tween solution, membranes were incubated with goat antirabbit Ig conjugated to horseradish peroxidase (Bio-Rad Laboratories, Inc., Marnes la Coquette, France) for 1 h at room temperature. Immune complexes were revealed by the ECL method using the enhanced chemiluminescent substrate from Amersham Pharmacia Biotech (Orsay, France) and exposed to Kodak X-OMAT AR films (Eastman Kodak Co., Rochester, NY). Membranes previously used to detect pendrin were incubated with a monoclonal antibody directed against the -subunit of Na⁺,K⁺-adenosine triphosphatase (Na⁺,K⁺-ATPase; final dilution, 1:25,000) and then with a biotinylated goat antimouse Ig from Amersham Pharmacia Biotech Immune complexes were visualized using streptavidin conjugated to alkaline phosphatase (from Amersham Pharmacia Biotech) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate as substrate. The monoclonal anti--Na⁺,K⁺-ATPase antibody developed by Douglas M. Fambrough was obtained from the Developmental Studies Hybridoma Bank maintained by Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine (Baltimore, MD), and Department of Biological Sciences, University of Iowa (Iowa City, IA). Western blot signals from the ECL (pendrin) and alkaline phosphatase (Na⁺,K⁺-ATPase -subunit) reactions were quantified using a two-step procedure. Images of films generated by the ECL reaction and images of Immobilon membranes after detection of the alkaline phosphatase activity were digitized using an Epson 1600Pro scanner (Seiko Epson Corp., Nagano-Ken, Japan) and the TWAIN Pro software with an 8-bit gray scale intensity and 300 pixels per inch spatial resolution. Quantification of signals was performed with Adobe Photoshop 5.0 software (Adobe Systems Inc., San Jose, CA), using the histogram function. The net average luminance of each signal, determined on a 256 gray level scale, was calculated as the difference between the average luminance in a window delimiting the spot and the average luminance in a window of the same size in a part of the film or membrane representative of the background. Results of independent Western blots were expressed in luminance values. To compare Western blot signals obtained from the large number of experiments required for the serial analyses in duplicate or triplicate of the 50 tissue samples, a NT membrane preparation (RP2) was included on each Western blot and used as an internal reference. Luminance values were expressed as a percentage of the value obtained with RP2.

Immunofluorescence labeling

Frozen thyroid tissue sections (6 µm) were fixed in 4% paraformaldehyde in PBS for 15 min. After washing in PBS, cryosections were incubated overnight at 4 C with 200 µg/ml IgG from pAb 827. Immune complexes were detected using a fluorescein isothiocyanate-labeled antirabbit antibody (from Sigma). After a 1-h incubation at room temperature, tissue sections were washed in PBS and observed using videomicroscope equipment previously described (13). Appropriate controls were performed in parallel to check autofluorescence and nonspecific reactions.

RNA extraction and detection of PDS and NIS (Na⁺/iodide sympoter) mRNA by RT-PCR

Total RNA was isolated from thyroid tissues using the phenol-chloroform extraction procedure according to Chomczynski and Sacchi (14). The RNA concentration was determined by absorbance measurements at 260 nm, and RNA purity and integrity were assessed by determination of the A260/A280 ratio and electrophoresis on 1% agarose, followed by ethidium bromide staining of 28S and 18S rRNA.

Total RNA (1 µg for PDS and 3 µg for NIS) was reverse transcribed in a 20-µl volume reaction using 200 U Moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, WI), 60 nmol of each dNTP (Life Technologies, Inc., Cergy Pontoise, France), 24 U ribonuclease inhibitor (Promega Corp.), and 100 pmol specific antisense primer: 5'-TAC GCA TAG CCT CAT CCT GGA CAT C-3' for PDS and 5'-GAG CCG CTA TAC ATT CTG GA-3' for NIS. Each sample was incubated for 1 h at 42 C, followed by 5 min at 95 C. PCR was carried out from 4 µl cDNA template solution (200 ng total RNA equivalent) for PDS and from 10 µl cDNA template solution (1500 ng total RNA equivalent) for NIS. The reaction mixture (100 µl) contained 2.5 U Taq DNA polymerase (Life Technologies, Inc.), 60 nmol of each dNTP, 100 pmol of the antisense primers described above, and 100 pmol of the sense primers: 5'-CAC AGT TGG ATT TGA TGC CAT TAG AGT A-3' for PDS and 5'-CTT CTG AAC TCG GTC CTC AC-3' for NIS in the buffer supplied with the enzyme. Samples were submitted to 35 cycles of amplification at 95 C for 1 min, 58 C for 1 min, and 72 C for 2 min, with a final extension at 72 C for 10 min. The resulting PCR products were submitted to electrophoresis in 2% agarose gels stained with 0.5 µg/ml ethidium bromide and photographed. The size of the expected fragment was 626 and 454 bp for PDS and NIS, respectively. The identity of the PDS and NIS PCR products was confirmed by Southern blot using specific probes previously tested for their ability to label the 5-kb PDS mRNA and the 3-kb NIS mRNA, respectively, by Northern blot.

Results

Characterization of pendrin expressed by human thyroid cells

The peptide (amino acids 766–780) corresponding to the C-terminal sequence of human pendrin, selected by antigenicity prediction analyses, coupled to keyhole limpet hemocyanin, has proved to be immunogenic. The selected immune serum, pAb 827, had a high antipeptide antibody titer in the ELISA; at dilutions up to 1 to 2 x 10⁶, the immune serum presented signals (A405 values) significantly higher than those obtained with the preimmune serum. The binding of antipeptide antibodies to the immobilized synthetic peptide in the ELISA was inhibited by a prior incubation with the free peptide. The peptide concentration that produced 50% inhibition was about 3 x 10^-7 M (data not shown). When tested on membrane fractions from different human thyroid tissue samples by Western blot under denaturing conditions, pAb 827 (at dilutions ranging from 1:200 to 1:2000) detected a single band migrating as a 110- to 115-kDa component (Fig. 1A). Although low, the background of the Western blot could be reduced by using the IgG fraction of pAb 827 instead of the complete immune serum. This fraction will be named pAb 827 IgG throughout the paper. Disulfide bond reduction of thyroid membrane proteins somewhat increased, and thermodenaturation slightly reduced pendrin immunoreactivity. However, none of the treatments, alone or in combination, affected the electrophoretic mobility of immunolabeled pendrin. As the molecular species immunodetected as pendrin had an apparent molecular mass higher than that predicted from the primary sequence, we examined whether the 110- to 115-kDa pendrin could correspond to a glycosylated protein. Treatment of thyroid membranes with N- glycosidase F, an enzyme that cleaves N-linked oligosaccharides, yielded an 85-kDa component, the size of which falls within the expected molecular mass for the pendrin polypeptide chain (Fig. 1C). The 85-kDa pendrin or deglycosylated form of pendrin was not detected in thyroid membrane fractions. As shown in Fig. 1D, the intensity of the pendrin signal, obtained with pAb 827 IgG, was proportional to the membrane protein input. Pendrin was generally detected from 10 µg membrane protein from normal thyroid tissue.

View larger version (32K): [in this window] [in a new window]   Figure 1. Characterization of human pendrin by Western blot. A, Immunodetection of pendrin in human thyroid tissues. Protein (30 µg) from membrane fractions prepared from three normal thyroid tissue samples (a–c) were subjected to PAGE in the presence of SDS and electrotransferred onto Immobilon P membrane. Transfer membranes were incubated with either pAb 827 or the preimmune serum at a 1:250 dilution. The position of proteins of known molecular mass (expressed in kilodaltons) is indicated on the left of the panel. The arrow indicates the mobility of the immunoreactive protein. B, Effect of disulfide bond reduction and thermodenaturation on the immunoreactivity of pendrin. Membrane proteins (30 µg) from a thyroid tissue sample were solubilized in the electrophoresis buffer containing SDS and incubated without (lanes 1 and 3) or with (lanes 2 and 4) 0.125 M dithiothreitol for 30 min at room temperature and then subjected (lanes 3 and 4) or not (lanes 1 and 2) to a 5-min treatment at 95 C. The resulting solutions were submitted to the Western blot procedure using the IgG fraction from pAb 827 (40 µg/ml). C, Evidence that pendrin is a glycoprotein. Samples of thyroid membrane proteins (40 µg) were incubated with increasing concentrations of N-glycosidase F in the electrophoresis buffer containing SDS for 3 h at 37 C and analyzed by Western blot as described above. Arrows indicate the glycosylated (110-kDa) and the deglycosylated (85-kDa) forms of human pendrin. D, Semiquantitative detection of pendrin by Western blot. Increasing amounts of membrane protein prepared from two normal human thyroid tissue samples were submitted to the Western blot procedure without prior disulfide bond reduction or thermodenaturation. ECL signals (inset) were scanned and quantified as reported in Materials and Methods. Amounts of immunoreactive pendrin, expressed in luminance values, are plotted as a function of the amount of membrane protein loaded in each lane.

View larger version (31K): [in this window] [in a new window]   Figure 2. Analysis of human pendrin by velocity sedimentation on sucrose gradient. Crude thyroid membranes (3 mg protein) were suspended in 1% Triton X-100 in 10 mM Tris, pH 7.4; allowed to stand for 45 min at room temperature; and centrifuged at 100,000 x g for 30 min at 4 C. Solubilized membrane proteins (2 mg) were loaded at the top of 5–20% sucrose gradients prepared in 10 mM Tris, pH 7.4, supplemented with 0.1% Triton X-100. Gradients were centrifuged at 100,000 x g for 16 h at 4 C using an SW41 rotor from Beckman Coulter, Inc. (Palo Alto, CA). Fractions (0.425 ml) collected from the top to the bottom of the gradients were assayed for pendrin and Na+,K+-ATPase -subunit by Western blot. Proteins (300 µg) of known molecular mass [BSA (67 kDa), aldolase (158 kDa), catalase (232 kDa), and apoferritine (440 kDa)] were centrifuged under the same conditions in separate gradients run in parallel. Protein profiles were determined by absorbance measurements at 595 nm using the Bradford reagent. Upper panels, Pendrin and Na+,K+-ATPase - subunit detection by Western blot. Middle panel, Distribution of immunoreactive pendrin and Na+,K+-ATPase -subunit along the gradient. Data (luminance values) are from quantitative analyses of Western blot signals. Lower panel, Sedimentation profiles of marker proteins.

View larger version (65K): [in this window] [in a new window]   Figure 3. Pendrin labeling on human thyroid cryosections using pAb 827 antipeptide antibodies. Cryosections prepared from normal thyroid tissue were fixed and incubated with 200 µg/ml pAb 827 IgG (A–C) or the corresponding preimmune serum at a 1:50 dilution (D). Immune complexes were detected using goat antirabbit Ig coupled to fluorescein isothiocyanate. Fluorescence was located at the apical domain of thyrocytes delimiting the follicle lumen (FL). Bars, 30 µm (A), 10 µm (B), and 3 µm (C and D).

Pendrin was assayed at the same time in tumor and paired NT samples from crude membrane fractions corresponding to the 100,000 x g pellet prepared from tissue homogenates. The data reported in Table 1 show that the total amount of protein per mass of tissue and the proportion of particulate or membrane protein (100,000 x g pellet) obtained from tumors (TA, FA, and PC) and paired NT samples were very similar. Protein of the crude membrane fractions represented 25–35% of total protein. The proportion of membrane protein obtained from PC samples was slightly higher than that of paired NT samples. This difference might be explained by the presence of a higher proportion of soluble TG in NT than in PC or, conversely, by a higher cellularity of tumors compared with NT.

View larger version (75K): [in this window] [in a new window]   Figure 4. Comparative analyses of the pendrin content of thyroid tumors and paired NT samples. Membrane fractions (30 µg protein) from thyroid tumors and paired NT samples were subjected to Western blot analysis using pAb 827 IgG and the ECL procedure. The -subunit of Na+,K+-ATPase was then detected on the same transfer membranes using a monoclonal antibody and the alkaline phosphatase-based colorimetric detection. Paired samples were analyzed simultaneously. Data from six representative paired samples are shown (one TA, one FA, and four PC) and their adjacent NT.

View larger version (16K): [in this window] [in a new window]   Figure 5. Semiquantitative estimates of pendrin and Na+,K+-ATPase -subunit contents of hyperfunctioning or hypofunctioning tumors and paired NT samples. Membrane fractions from 25 tumors (3 TA, 4 FA, and 18 PC) and the 25 paired NT samples were analyzed for their pendrin (upper panel) and Na+,K+-ATPase -subunit (lower panel) contents by Western blot. Each of the 50 samples was subjected to 2–3 pendrin and Na+,K+-ATPase -subunit determinations on separate Western blots. An internal reference (RP2), the membrane fraction from a normal thyroid tissue sample, was included on each Western blot membrane to correct for variations in signal amplitude between Western blots. Western blot signals were quantified as described in Materials and Methods. Luminance values were expressed as a percentage of RP2 value. Columns and vertical bars represent the mean ± SEM of the values obtained with NT paired to TA and TA (n = 3), NT paired to FA and FA (n = 4), and NT paired to PC and PC (n = 18). a, P < 0.05;b, P < 0.01.

We further investigated the alterations of pendrin expression in hypofunctioning tumors by analyzing the PDS transcript level in 10 of the 22 tumor/paired NT samples that were available in duplicate for RNA extraction. PDS transcripts were amplified by RT-PCR. NIS transcripts, which are known to be decreased by more than 2 orders of magnitude in both PC and FA, were amplified in parallel from the same RNA preparations. The PDS and NIS RT-PCR procedures were based on the use of specific primers to synthesize cDNAs. It was verified that PDS and NIS amplicons were generated in amounts proportional to the logarithm of the initial PDS or NIS cDNA concentration. The results of amplification of PDS and NIS transcripts from total RNA of a PC and its paired NT are shown in Fig. 6, A and B; the PDS transcript content of the tumor was about 3 times lower than that of the NT. NIS transcripts were amplified in NT, but remained undetectable in the tumor even at the highest cDNA concentration (corresponding to 1.5 µg RNA retrotranscribed). Amplification of PDS and NIS transcripts was carried out on 10 paired samples (6 PC/NT and 4 FA/NT). Representative results are reported in Fig. 6C. PDS transcripts were amplified in all samples; the amount of PDS amplicons generated from tumor RNA was similar to that obtained from NT RNA in 4 of the 6 PC and 3 of the 4 FA. In 2 PC and 1 FA, PDS amplicons were decreased by 20–80% compared with NT. RT-PCR data were in agreement with pendrin measurements by Western blot. Indeed, in the 7 tumors with a PDS transcript level indistinguishable from that of paired NT, the average tumor pendrin content (expressed as a percentage of the NT value) was close to 100%. In the 3 other tumors, the pendrin content of the tumor was reduced by about 40%. NIS transcripts were detected in the 10 NT, but were not amplified in the tumors, except in 1 PC in which NIS amplicons were found in trace amounts.

View larger version (21K): [in this window] [in a new window]   Figure 6. RT-PCR analyses of the PDS and NIS transcript content of hypofunctioning tumors and paired NT samples. A and B, Validation of the RT-PCR assay conditions of PDS and NIS transcripts. RNA from a PC and its paired NT was retrotranscribed as indicated in Materials and Methods using 1 and 3 µg total RNA for PDS and NIS cDNA synthesis, respectively. PCR was carried out from serial dilutions (1:2 to 1:200) of the cDNA solutions. After 35 cycles of amplification, equal volumes (10 µl of 100 µl) of the resulting PCR solutions were analyzed on 2% nondenaturing agarose gel. PCR amplification products (PDS or NIS amplicons) were detected by ethidium bromide staining and photographed. The gels were scanned, and signals were quantified using Adobe Photoshop software and expressed in arbitrary units (a.u.). The results of PDS and NIS transcript amplification from the NT sample () and the tumor () are presented in A and B, respectively. No NIS amplicon was observed for the tumor, even at the lowest dilution of the cDNA solution corresponding to 1500 ng total RNA retrotranscribed. C, Comparative amplification of PDS and NIS transcripts in tumors and paired NT samples. PCR was performed from cDNA solutions corresponding to 200 and 1500 ng total RNA retrotranscribed for PDS and NIS, respectively. Other conditions were the same as those mentioned above. The ethidium bromide-stained PDS and NIS amplicons obtained from five tumors (two FA and three PC) and paired NT samples are presented in the upper and lower panels, respectively. N, Normal tissue; T, tumor; C- and C+, negative and positive PCR controls, respectively.

Since the cloning of the PDS gene, pendrin, its product, has been subjected to numerous studies, mainly aimed at the definition of its functional activity. Surprisingly, the basic characteristics of pendrin expressed in the thyroid or kidneys of laboratory animals or humans are not known. Using antipeptide antibodies directed against the last 15 amino acids of the human pendrin sequence, we show that pendrin, expressed in human thyroid cells, has an apparent molecular mass of 110–115 kDa by SDS-PAGE analysis. The difference between the molecular mass of immunoreactive polypeptide and the predicted molecular mass of pendrin (86 kDa) is mainly due to its posttranslational modification by glycosylation. Indeed, under the action of N-glycosidase F, the molecular mass of immunoreactive pendrin shifted to about 85 kDa. This finding is in agreement with the existence of several potential N-glycosylation sites located on the first and second extracellular loops according to the pendrin model with 11 transmembrane domains proposed by Everett et al. (3) from structure prediction analyses. The actual size of the mature protein is different from that previously reported for the protein synthesized by cells transfected with the PDS cDNA (8), which was close to 95 kDa. We never observed immunoreactive pendrin species of high molecular mass, which is at variance with the study by Royaux et al. (8) based on analyses of the recombinant protein. As expected for a glycoprotein, immunoreactive pendrin migrated as a rather large band in SDS-PAGE.

In nondenaturing conditions, Triton X-100-solubilized pendrin had an apparent molecular mass substantially higher than that determined by SDS-PAGE. This increase in molecular mass is probably due to the binding of a large number of detergent molecules to the numerous hydrophobic domains of pendrin. Sucrose gradient centrifugation studies suggest that pendrin is mainly monomeric; the fastest sedimenting pendrin species probably correspond to oligomers either preexisting in native plasma membranes or artifactually formed during detergent solubilization. It is noteworthy that under the same detergent treatment conditions, the Na⁺,K⁺-ATPase composed of stoichiometric amounts of - and ß-subunits (15) sedimented as expected as an approximately 150- to 170-kDa species with a minimal amount of material of higher size. As previously reported by others (8, 12), immunoreactive pendrin was located at the apical plasma membrane of human thyrocytes. The immunolabeling was rather homogeneous, suggesting that in the normal gland most thyroid follicular cells probably express pendrin. Accordingly, pendrin was readily detected by Western blot in all 25 NT samples we examined.

From semiquantitative estimates of the tissue pendrin content, we identified a significant reduction of pendrin in NT paired to TA compared with other NT. As plasma TSH was decreased in patients with TA, the decrease in pendrin in NT paired to TA might be related to the reduction of thyroid cell stimulation by TSH. However, previous studies of PDS gene expression by FRTL-5 cells (8) have clearly shown that PDS mRNA levels remain unchanged in response to TSH treatment. These data and the fact that another membrane protein, the Na⁺,K⁺-ATPase -subunit, was also decreased in NT paired to TA suggest that the change in pendrin expression may result from a general decrease in thyroid cell activity rather than from a specific change in PDS gene expression related to the decrease in plasma TSH concentration. The observation of a similar pendrin content in TA and NT coming from euthyroid patients (NT paired to either FA or PC) would indicate that activation of the cAMP cascade in TA does not have an impact on pendrin expression.

As hyperfunctioning (TA) and hypofunctioning (FA) benign tumors exhibit a very similar pendrin content, pendrin expression does not seem to be linked to the iodine accumulation and utilization activities of the thyroid tissue. The pendrin content of the 18 malignant thyroid tumors we analyzed was, on the average, not greatly altered compared with that of paired NT. It was significantly decreased in about half of the cases. We did not identify any relationship between the pendrin content of PC and the tumor staging. The complementary RT-PCR analyses also showed no or limited alterations of the PDS transcript levels in FA and PC. By comparison, the NIS transcript levels of the same tumors (PC and FA) were decreased by more than 2 orders of magnitude compared with that of paired NT. These data indicate that the mechanisms governing the expression of the PDS gene and that of the NIS gene are completely distinct.

The elevation of Na⁺,K⁺-ATPase -subunit content in the different types of thyroid tumors (compared with paired NTs) might be related to a general increase in metabolic activities of tumor cells compared with normal cells. Indeed, Na⁺,K⁺-ATPase, by establishing and maintaining Na⁺ and K⁺ gradients between cells and the extracellular fluid through its -subunit, is involved in many different cellular processes. There are numerous reports showing that enzyme activities involved in various metabolic pathways, including phosphofructokinase (16), pyruvate kinase (17), thymidylate synthase, thymidine kinase (18), and cathepsins (19), are markedly increased in thyroid tumors (mainly carcinomas). In the first two above-mentioned reports, there was a relationship between the increase in enzyme activity and the proliferative activity of tumors; the higher the specific activity of the enzyme, the higher the proliferative index. The difference in Na⁺,K⁺-ATPase -subunit content between normal and tumoral tissue samples might thus indicate a difference in cell proliferation activity. Thus, the Na⁺,K⁺-ATPase -subunit tissue content appears as a parameter validating tissue sampling and classification and as an internal reference for interpreting variations in the expression levels of pendrin.

As pendrin is capable of transporting iodide among various other anions and is located at the apical plasma membrane of thyrocytes (9, 10), it has been postulated that this protein might represent the transporter of iodide from the cells to the lumen of thyroid follicles. At present, there is no direct experimental evidence supporting this hypothesis. Analyses of the changes in pendrin expression pattern in relation to changes in iodine handling capacity of the thyroid tissue, such as those we reported here, generate indirect information about the involvement of pendrin in thyroid iodine fluxes and metabolism. Our data show that the pendrin content of thyroid tissue does not vary, or varies only moderately, with the functional activity of the gland in terms of iodine accumulation and utilization assessed by scintigraphy. Thus, if pendrin plays a role as an apical iodide transporter, alteration of its expression level should not be a causal factor for decreasing the capacity of hypofunctioning benign or malignant tumors to accumulate and use iodine.

Acknowledgments

We thank Profs. F. Berger, J. Orgiazzi, M. Pugeat, and J.-P. Riou; Drs. M.-H. Bernard, H. Bornet, F. Borson-Chazot, B. Hughes, and G. Sassolas; and members of the organizing and supervision committees of the Lyon Thyroid Tumor Bank for their contributions to this study.

Footnotes

This work was supported by a grant from the Programme Hospitalier de Recherche Clinique Cancer Différencié de la Thyroide, 1998–2001 and Contract N01-HD-2–3144 from the NICHHD (to Department of Biological Sciences, University of Iowa).

Abbreviations: FA, Follicular adenoma; Na⁺,K⁺-ATPase, Na⁺,K⁺-adenosine triphosphatase; NIS, Na⁺/iodide symporter; NT, normal tissue; PC, papillary carcinoma; PDS, Pendred syndrome; TA, toxic adenoma.

Received September 19, 2001.

Accepted December 26, 2001.

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