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Endocrinology Vol. 148, No. 4 1754-1763
Copyright © 2007 by The Endocrine Society

Proteomic Profiling of Cold Thyroid Nodules

Kerstin Krause, Stefan Karger, Angelika Schierhorn, Sylvie Poncin, Marie-Christine Many and Dagmar Fuhrer

Medical Department III (K.K., S.K., D.F.), University of Leipzig, D-04103 Leipzig, Germany; Research Unit Enzymology of Protein Folding (A.S.), Max-Planck Society, D-06120 Halle/Saale, Germany; and Department of Histology (S.P., M.-C.M.), Universite Catholique de Louvain Medical School, B-1070 Brussels, Belgium

Address all correspondence and requests for reprints to: Dagmar Fuhrer M.D., Ph.D., Medizinische Klinik III, Universität Leipzig, Ph.-Rosenthal-Strasse 27, D-04103 Leipzig. E-mail: fued{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cold thyroid nodules (CTNs) represent a frequent endocrine disorder accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. Benign CTNs need to be distinguished from thyroid cancer, which is relatively rare. The molecular etiology of benign CTNs is unresolved. To obtain novel insights into their pathogenesis, protein expression profiling was performed in a series of 27 solitary CTNs (10 follicular adenoma and 20 adenomatous nodules) and surrounding normal thyroid tissues using two-dimensional gel electrophoresis combined with mass spectrometry analysis, Western blotting, and immunohistochemistry. The proteome analysis revealed a specific fingerprint of CTNs with up-regulation of three functional systems: 1) thyroid cell proliferation, 2) turnover of thyroglobulin, and 3) H2O2 detoxification. Western blot analysis and immunohistochemistry confirmed the proteome data and showed that CTNs exhibit significant up-regulation of proteins involved in thyroid hormone synthesis yet are deficient in T4-containing thyroglobulin. This is consequential to intranodular iodide deficiency, mainly due to cytoplasmic sodium iodide symporter localization, and portrays the CTN as an activated proliferating lesion with an intranodular hypothyroid milieu. Furthermore, we provide preliminary evidence that up-regulation of H2O2 generation in CTNs could override the antioxidative system resulting in oxidative stress, which is suggested by the finding of raised 8-oxo-guanidine DNA adduct formation in CTNs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COLD THYROID NODULES (CTNs) are characterized by a scintiscan appearance of decreased or lack of radionuclide uptake when compared with the surrounding normal thyroid tissue (ST). CTNs represent a frequent endocrine disorder, accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. The main clinical concern with CTNs is that of underlying malignancy, because thyroid cancer usually presents as a hypofunctional lesion. However only 5–10% of CTNs actually represent thyroid malignancy (1, 2).

With the advent of molecular biology, specific genetic alterations have been identified in several thyroid cancer types, notably RET-PTC and BRAF V600E in papillary thyroid cancer, PAX8-PPAR{gamma}1 in subsets of follicular thyroid cancer, and RET-protooncogene mutations in medullary thyroid cancer (3, 4, 5, 6). In contrast, the molecular pathogenesis of benign CTN is still unresolved. Several candidate genes such as NIS, TPO, RAS, and EPAC have been investigated, but no specific mutations have been identified (7, 8, 9, 10, 11).

Hence, we decided to apply proteomics to define a characteristic protein fingerprint of CTNs as a prerequisite for additional insights into their pathogenesis. With the possibility for concomitant detection of thousands of proteins, proteomics has become an important screening tool for the identification of differential protein expression patterns that occur with, for example, altered cell function/signal transduction (12). Using a combined approach of two-dimensional gel electrophoresis (2D-GE) and mass spectrometry (MS) followed by Western blot analysis and immunohistochemical studies, we demonstrate for the first time that benign CTNs are characterized by the up-regulation of several proteins involved in thyroid hormone synthesis, with a strong induction of the H2O2-generating and detoxifying systems. Moreover, increased formation of 8-oxo-guanidine DNA adducts was detected in CTNs and could besides the increased cell proliferation in these nodules confer an increased potential for genotoxicity and mutagenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental subjects
Samples from 27 patients undergoing surgery for a solitary benign CTN were studied (Table 1Go). Thyroid nodules were identified by ultrasound, corresponding scintiscan result of a hypofunctioning nodule, and intraoperative inspection confirming the presence of a solitary thyroid lesion. Immediately after surgical removal, the native tissue was inspected by the pathologist and tissue sections were shock-frozen in liquid nitrogen and prepared for histological analysis, respectively. Classification of the thyroid nodules was performed by the pathologist according to World Health Organization criteria. The term follicular adenoma was applied for benign follicular lesions, which were completely encapsulated, and the term adenomatous nodule (colloid nodule) was applied to benign lesions, which were only partly encapsulated. The surrounding thyroid tissue was investigated histologically and showed normal thyroid morphology in all patients. All patients were euthyroid, and none of the patients received medical therapy (levothyroxine or iodine). In addition, presence of thyroid autoantibodies [thyroid peroxidase (TPO), thyroid-stimulating receptor, and thyroglobulin (Tg) antibodies] was excluded in all patients. All patients gave informed consent, and the study was approved by the local ethics committee.


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  		TABLE 1. Clinical details of analyzed tissue samples

 
For the proteome analysis and Western blotting, 20 CTNs (seven follicular adenomas and 13 adenomatous nodules) and corresponding ST of the same patients were studied. Twenty samples were investigated by immunohistochemistry (IHC) with an overlap of 13 of 20 samples investigated by 2D-GE, Western blotting, and IHC (Table 1Go).

Protein extraction and determination of protein content
Cytosolic protein extracts were prepared as previously described (13, 14). Briefly, frozen thyroid samples (–80 C) were transferred into a cooled mortar (–196 C). The tissue was ground with Tris buffer (50 mM Tris, 100 mM KCl, and 20% glycerol; pH 7.1) in the presence of protease inhibitors (one tablet of Complete Protease Inhibitor Cocktail per 2 ml Tris buffer, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of pepstatin A and leupeptin; all inhibitors were from Roche Diagnostics GmbH, Mannheim, Germany). The samples were thawed and centrifuged at 100,000 x g for 1 h at 4 C. Subsequently, the supernatant was shock-frozen in liquid nitrogen and stored at –80 C.

Protein content of all samples was measured using the BCA kit (Pierce, Milwaukee, WI). BSA was used as standard. Thyroglobulin measurement was performed using the Brahms Tg RIA kit (Brahms AG, Hennigsdorf, Germany).

2D-GE
Proteins were precipitated in 20% trichloroacetic acid/acetone, and pellets were afterward solubilized in isoelectric focusing buffer containing 9.5 M urea, 2 M thiourea, 4% CHAPS, 50 mM dithiothreitol, and 0.5% immobilized pH-gradient (IPG) buffers. All reagents were obtained from Amersham Biosciences (Freiburg, Germany). Isoelectric focusing was performed in 24-cm-long pH 4–7 IPG strips (Amersham Biosciences) with 1000 µg protein load per strip until 84 kVh was reached. Equilibrated strips were placed on top of 8–16% SDS-polyacrylamide gels, and electrophoresis was performed at 10 C at a constant current of 60 mA per gel (14). Triplicates of cytosolic protein extracts of the CTN (3 x 20 CTNs) and the corresponding normal thyroid tissue of the same patients (3 x 20 normal thyroids) were run in parallel through both electrophoresis steps.

Protein detection and spot analysis
The fluorescent ruthenium II Tris bathophenanthroline disulfonate (RuBPS) stain was used for quantitative protein detection. RuBPS was synthesized according to Rabilloud et al. (40), and staining was performed as described (13).

Gels were scanned at 100 µm resolution using the Molecular Imager FX System (Bio-Rad Laboratories, Munich, Germany). Fluorescence image acquisition of RuBPS-stained gels was performed with a 480-nm laser. Spot detection and normalization was carried out with the PDQuest software (version 7.2; Bio-Rad Laboratories). One of the triplicate gels of the respective ST was chosen as a reference gel. Normalization of spot quantities was performed by dividing the raw quantity of each spot in the gels by the total quantity of all spots included in the reference gel. When comparing nodular and normal tissues, ratios of the normalized quantity values from normal vs. nodular spots were compared with each other, and a mean difference in spot quantity was calculated. The two-sided t test was used to determine significant differences in protein expression among CTNs and the corresponding normal thyroid tissues. A P value of <0.05 was considered statistically significant.

For spot picking, the RuBPS gels were restained with colloidal Coomassie blue after scanning (13).

Protein identification
Protein spots were cut out and washed three times with water, twice with 50 mM ammonium bicarbonate, and finally with 50 mM ammonium bicarbonate/50% acetonitrile. Gel pieces were dried, reswollen in 20 µl 50 mM ammonium bicarbonate (pH 8.0), and digested with trypsin (Promega, Madison, WI) overnight at 37 C. Tryptic peptides were extracted from gel pieces and desalted online through a 300-µm by 5-mm C18 trapping cartridge (Dionex GmbH, Idstein, Germany) at a flow rate of 20 µl/min for 5 min. Before MS, peptides were separated on a 750-µm by 15-cm, 3-µm C18 100-Å PepMap column (Dionex). MS/MS analysis was performed on a Q-TOF 2 mass spectrometer (Micromass, Manchester, UK) equipped with a modified nano-ESI. Spectra were acquired in MS mode at 1 sec/scan and in MS/MS mode at 3 sec/scan. Database searches were performed using the Mascot software (Matrix Science, London, UK) (http://www.matrixscience.com).

Western blot analysis
Protein lysates (100 µg), adapted to an equal Tg amount, were separated on 12 and 8% SDS gels for cathepsin B (CB) and amyloid precursor protein (APP), respectively (14). For Tg immunoblotting, the protein load was adjusted to 10 µg Tg per lane and separated on 4–8% and 4–16% SDS gradient gels. The following antibodies were used: anti-APP (22C11; Chemicon, Hampshire, UK), anti-CB (Merck Biosciences, Bad Soden, Germany), and anti-Tg (Labvision, Fremont, CA). Blots were probed overnight with primary antibodies diluted (1:1000 for Tg and CB and 1:500 for APP) in Tris-buffered saline (10 mM Tris, pH 8.0, and 150 mM NaCl) and 5% BSA. Blots were then reprobed with secondary antibodies coupled to horseradish peroxidase at a dilution of 1:10,000 (Cell Signaling, Charlottesville, VA) and visualized by enhanced chemiluminescence (Pierce).

IHC
The 3-µm thyroid tissue sections were prepared and subjected to IHC using the protocols described by Many and colleagues (15). The following antibodies were used: Tg (Dako, Glostrup, Denmark), Tg-I (B1, monoclonal antibody) (16), sodium iodide symporter (NIS) (monoclonal antibody) (17), thyroid oxidases (ThOXs) (polyclonal antibody raised against ThOX1 and ThOX2) (18), TPO (monoclonal antibody mAb47) (19), and peroxiredoxin 5 (PDRX-5) (20). 8-Oxo-guanidine labeling was performed using an 8-deoxyguanine antibody (Chemicon) and the Dako LSAB+System-AP kit according to the manufacturers’ instructions.

Semiquantitative analysis of staining intensity for both cold nodules and adjacent tissue was performed according the following procedure: 15 visual fields (x400 magnification) were counted with five follicles per visual field. For each sample, the percentage of positive stained cells per follicle was determined. The analysis was performed by two independent investigators (S.K. and K.K.). Furthermore, the SEM between all 20 investigated tissue sections (nodule or normal tissue) was determined.

The statistical significance of the percentage of positively stained cells in CTNs vs. STs was tested by two-sided ANOVA.

Endoglycosidase H (Endo H) treatment
Ten micrograms Tg were digested with 0.3 mU/liter Endo H (Merck, Darmstadt, Germany) in digestion buffer containing 250 mM sodium citrate (pH 5.3), 2.5% SDS, 5% mercaptoethanol, and 50 mM EDTA for 15 min at room temperature. Subsequently, SDS-sample buffer was added, and samples were heated to 95 C. Electrophoresis was carried out on 6% SDS gels. Western blotting with a monoclonal anti-Tg antibody was performed as described above.

Sequencing of Tg gene
For sequence analysis of the Tg gene, cDNAs were prepared from four CTNs and their corresponding surrounding thyroid tissues (previously analyzed by 2D-GE). The coding Tg gene sequence was amplified by PCR using 20 overlapping PCR primer pairs (available on request). Polyethylene-glycol-purified PCR products were sequenced using BigDye terminator sequencing chemistry (Applied Biosystems, Foster City, CA), and sequence analysis was performed on the Genetic Analyzer ABI 377 (Applied Biosystems) as described (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proteome analysis provides evidence for differential regulation of three functional systems in CTNs
We first applied a proteomics screening approach involving 2D-GE to define a specific differential protein expression pattern of benign CTNs. A series of 20 solitary benign CTNs (seven follicular adenomas and 13 adenomatous nodules) were investigated in comparison with their respective normal ST. Triplicates of cytosolic protein extracts of nodular and normal thyroid tissues of the patient sample were run in parallel throughout both electrophoresis steps, resulting in highly reproducible 2D patterns with intraassay correlation coefficients of r = 0.94 and r = 0.90, respectively. Approximately 1500 protein spots were detected per 2D gel (pH 4–7, 24-cm IPG strips; Fig. 1Go). To assess quantitative and qualitative alterations in protein expression, 2D gels were stained with the fluorescent RuBPS stain and were analyzed using the PDQuest 7.2 software. The criteria for spot selection were a relative difference in spot intensity (CTN vs. ST) of at least a factor of 2. Table 2Go shows an extract of differentially expressed protein spots (n = 157) between CTNs and STs, which were subsequently identified by MS. A consistent expression pattern (≥10 of 20 CTNs, ≥2-fold difference in spot quantity) was detected for 11 proteins, whereas novel protein spots, which are exclusively expressed in either tissue, were found to correspond to either Tg fragments (14 of 20) or CB isoforms (16 of 20 CTNs; Fig. 1Go and Table 3Go).


Figure 1
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  		FIG. 1. Representative 2D gel of cytosolic proteins of a CTN (no. 4; follicular adenoma). Protein (1000 µg) was separated on 24-cm pH 4–7 IPG strips and 8–16% SDS gels. Numbers mark location of differentially expressed protein spots in CTNs (Table 3Go).

 

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  		TABLE 2. Protein spots with differential expression between CTNs and corresponding ST

 

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  		TABLE 3. Proteins with consistent up-regulation in CTNs compared with the corresponding ST

 
These identified proteins, which are more abundant in CTNs, can be assigned to three functional systems: 1) cell proliferation [serum amyloid P-component, amyloid-ß-A4 binding protein (APP-BP)], 2) Tg processing [Tg fragments, CB, protein disulfide isomerase (PDI), heat-shock protein 90ß (HSP90ß), and calreticulin), and 3) the H2O2-detoxifying system [PDRX-2 and -6, glutathione S-transferase {pi} (GST-{pi}), and DJ-1].

Subsequently, the differential regulation of these functional systems was investigated in detail.

Up-regulation of the APP in CTNs
A fragment of the serum APP, the serum amyloid p-component, was significantly up-regulated in 17 of 20 CTNs (P < 0.01; Fig. 2AGo and Table 3Go). In addition, up-regulation of the APP-BP was detected in 13 of 20 CTNs (P < 0.03; Fig. 2BGo and Table 3Go). The serum APP, with isoelectric point (pI) of 6.5 (130 kDa) was not detectable due to the high abundance of Tg around this region (Fig. 1Go). The APP has recently been identified as a novel growth factor in thyroid epithelial cells. Cellular APP expression correlates with increased cell proliferation (22). Moreover, thyrocytes can secrete an N-terminal soluble fragment of APP, which can stimulate growth in an auto- and paracrine manner (22).


Figure 2
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  		FIG. 2. A and B, Gel sections demonstrating differential protein expression in a CTN (left) compared with surrounding normal thyroid tissue (right) of the same patient for serum amyloid P-component (A) and APP-BP (B); C, Western blot analysis confirming the higher expression of APP in CTNs compared with STs of the same patient (shown here for eight of 20 samples). Protein loads were normalized to 10 µg Tg per sample.

 
Additional evidence for differential APP expression in CTNs was provided by Western blot analysis. Two major bands were detected on 8% SDS gels corresponding to the mature O-glycosylated form of APP (130 kDa) and to the immature form of APP (110 kDa). Both were overexpressed in CTNs compared with their STs (Fig. 2CGo).

CTNs are marked by an altered Tg turnover
CB is overexpressed in CTNs.
A remarkable variation in protein expression was observed for CB and/or CB isoforms in 2D gels of 16 of 20 CTNs (Fig. 3AGo and Table 3Go). CB is relevant to apical Tg proteolysis resulting in the release of iodothyronines and iodotyrosyls. Subsequent immunoblots on 10% SDS gels using an anti-CB antibody revealed two major bands at 27 and 50 kDa corresponding to the mature form and immature form of CB, respectively. The 27-kDa mature and proteolytically active form was overexpressed in CTNs (Fig. 3BGo), which confirms results obtained by 2D-GE analysis.


Figure 3
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  		FIG. 3. A, Gel sections demonstrating differential protein expression of CB in a CTN (left) compared with ST (right) of the same patient; B, Western blot analysis confirming the higher expression of CB in CTNs compared with the STs of the same patient (shown here for eight of 20 samples). Protein loads were normalized to 10 µg Tg per sample.

 
Altered Tg expression in CTNs.
Eight protein spots, which were exclusively detected on 2D gels of 14 of 20 CTNs, were identified as Tg components. These Tg forms varied considerably in their pI and molecular weight (MW) and thus differed significantly from the calculated pI and MW of mature Tg (Table 4Go).


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  		TABLE 4. Tg components detected exclusively in CTNs

 
Immunoblots of Tg on 12% SDS gels and 4–8% SDS-gradient gels confirmed the higher expression of several Tg peptide fragments in CTNs compared with STs. Tg immunoreactivity was observed particularly in high-MW aggregates (>300 kDa) as well as in broad bands in the resolving gel. A characteristic band around 90 kDa was up-regulated in CTNs and a 75-kDa band was exclusively expressed in CTNs, whereas a band around 18 kDa was weakly expressed in CTNs compared with STs (data not shown).

Absence of T4-rich Tg in CTNs.
Because alterations in Tg expression in CTNs might lead to an impaired thyroid hormone formation in CTNs, IHC was applied to determine the presence of highly iodinated T4-rich Tg. For this, a monoclonal antibody directed to a specific hormonogenic region of the Tg N terminus in addition to a polyclonal anti-Tg antibody were applied to paraffin-embedded sections of CTNs and STs (15, 16). Importantly, although there was strong, equivocal Tg staining both in the follicular lumina and thyrocytes in all CTNs and STs (Fig. 4AGo), no staining was observed with the antibody specific for iodinated T4-rich Tg in any of the CTNs (Fig. 4BGo). In contrast, all corresponding STs exhibited positive endoluminal T4-Tg staining (Fig. 4BGo). This striking black and white T4-Tg image suggests a status of inefficient thyroid hormone synthesis in CTNs.


Figure 4
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  		FIG. 4. Immunohistochemical analysis of Tg and T4-rich Tg. A, Cytosolic and endoluminal expression of Tg in CTNs and STs; B, absence of T4-rich Tg in follicle cells and lumina of CTNs in contrast to T4-rich Tg in STs. Magnification, x400.

 
Neither a defective intracellular Tg transport nor mutations in the Tg gene are responsible for thyroid hormone deficiency in CTNs.
Three molecular chaperones were identified by proteome analysis to be differentially regulated in CTNs (calreticulin, PDI, and HSP90ß) (Fig. 5AGo). Calreticulin was overexpressed in 17 of 20 CTNs (P < 0.02; Table 3Go and Fig. 5AGoa, whereas PDI and HSP90ß were up-regulated in 16 of 20 and 14 of 20 CTNs compared with their ST (P < 0.02 and P < 0.03; Table 3Go and Fig. 5AGo, b and c, respectively).


Figure 5
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  		FIG. 5. A, Gel sections demonstrating differential expression of molecular chaperones in a CTN compared with ST of the same patient: a, calreticulin; b, PDI; c, HSP90. B, Gel sections showing differential expression of antioxidant proteins in a CTN compared with ST of the same patient: a, GST-{pi}; b, PDRX-2; c, PDRX-6; d, DJ-1.

 
These chaperones assist in part in the folding and assembling of Tg, while exerting a quality control in the endoplasmic reticulum (ER) (23, 24, 25, 26, 27, 28). It has been shown that the accumulation of misfolded Tg molecules induces the expression of ER chaperones, which bind to the misfolded Tg, thereby preventing its transition into the Golgi apparatus. Thus, we asked whether the induction of the respective proteins could be related to an abnormal Tg integrity, e.g. by altered posttranslational modifications. Therefore, protein extracts of CTNs and corresponding STs were treated with Endo H. Endo H digests only the high-mannose-type (ER-type) oligosaccharides. However, in all analyzed samples, exclusively Endo H-resistant bands were detected, arguing against a failure in the intracellular transport of Tg from the ER to the Golgi complex (data not shown).

In addition, to exclude Tg gene abnormalities, the entire coding region of the human Tg gene (8307 bp) was sequenced in four of 14 CTNs, in which short Tg forms were detected by 2D-GE, and their ST. Except for several base exchanges corresponding to silent mutations or known Tg polymorphisms, no somatic Tg mutations were found (data not shown).

CTNs exhibit a strong up-regulation of the antioxidative defense system
Proteome analysis showed a 5.0-fold overexpression of GST-{pi} in 15 of 20 CTNs compared with STs (P < 0.01; Fig. 5BGoa and Table 3Go). GST-{pi} is relevant for the reduction (detoxification) of hydrogen peroxide, which is produced by the ThOX1/2 system and is required by the TPO for iodination of Tg (18). Moreover, the antioxidant proteins PDRX-2 and -6 were significantly up-regulated in CTNs (3.3-fold, P < 0.03 for PDRX-2, and 2.8-fold, P < 0.02 for PDRX6; Fig. 5BGo, b and c, respectively, and Table 3Go).

In addition, up-regulation of DJ-1, a recently identified protein suggested to be involved in antioxidative defense, was detected by proteome analysis in 12 of 20 CTNs (3.2-fold; Fig. 5BGod and Table 3Go). IHC confirmed the significant up-regulation of cytosolic localized peroxidredoxins in CTNs compared with STs (78 ± 2.5 and 22 ± 2.5%, respectively; Fig. 6AGo).


Figure 6
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  		FIG. 6. IHC confirms the up-regulation of PDRX-5 in the cytoplasm of CTNs compared with STs (A) and the overexpression of NIS in CTNs vs. STs (B), which is mostly due to the intracellular NIS localization in CTNs (a) compared with the basolateral localization in STs (b). TPO (C) and ThOX1/ 2 (D) are overexpressed in CTNs vs. STs. Magnification, x400.

 
Because the proteome data strongly suggested alterations in thyroid hormone production in CTNs, we decided to perform a systematic investigation of the various components of the thyroid hormone synthesis system in these nodules.

IHC confirms intracellular NIS localization and reveals up-regulation of other essential components of the thyroid-hormone-producing system in CTNs
Two patterns of NIS expression were observed in our series of CTNs. First, up-regulation of NIS, which is responsible for iodide uptake into the thyroid cell, was detected in 14 of 20 CTNs compared with their STs (65.2 ± 3.5 vs. 45.9 ± 1.3% positively stained cells; Figs. 6BGo and 7Go). Therefore, NIS expression in CTNs was predominantly intracellular in contrast to the normal basolateral staining in the STs (see enlargements in Fig. 6BGo, a and b). Second, down-regulation of NIS with faint basolateral expression was found in six of 20 CTNs compared with their STs. Intracellular location or down-regulation of NIS has previously been reported by several authors in both thyroid malignancies and benign CTNs (9, 10, 29, 30, 31, 32).


Figure 7
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  		FIG. 7. Semiquantitative analysis of protein expression studied by immunohistochemistry (x400 magnification). Increased expression of essential enzymes involved in thyroid hormone synthesis occurred in 16 CTNs vs. ST (TPO, ThOXs, PDRX5, and NIS). The results are expressed as mean ± SEM. ***, P < 0.001 for CTNs vs. STs. No difference in Tg expression but complete absence of T4-Tg was noted in CTNs compared with STs.

 
Expression of pendrin, involved in apical iodine transport, was weak and restricted to the apical membrane in CTNs and ST. There was no significant difference in pendrin expression between CTNs and STs (data not shown).

In contrast, the TPO was strongly up-regulated in all CTNs vs. STs (85 ± 3.5 and 35 ± 3.4%, respectively; P < 0.001; Figs. 6CGo and 7Go). Its expression was limited to the apical membrane. In addition, we found a marked up-regulation of dual oxidases ThOX1 and ThOX2 in CTNs compared with ST (68.8 ± 2.7 and 15 ± 2.5%, respectively; P < 0.001; Figs. 6DGo and 7Go). ThOXs are essential components for the intrathyroidal generation of H2O2, which is a rate-limiting step in thyroid hormone synthesis.

Evidence for increased oxidative stress in CTNs
Because of the detection of the high expression of both H2O2-synthesizing proteins (ThOXs) and H2O2-detoxifying proteins (GST-{pi} and PDRXs), we asked whether the increased H2O2 generation in CTNs is sufficiently counterbalanced by up-regulation of the antioxidative system or causes oxidative stress. As an indirect indicator of increased oxidative stress, the formation of 8-oxo guanidine DNA adducts was investigated. Using a specific 8-oxo-guanidine antibody, we observed an increased staining for 8-oxo-guanidine DNA adducts in CTNs compared with their STs in CTNs with the most prominent staining occurring in microfollicular structures (Fig. 8Go).


Figure 8
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  		FIG. 8. A, Immunohistochemical analysis confirms the increased formation of 8-oxo-guanidine DNA adducts in CTNs compared with ST (x400 magnification); B, enlarged 8-oxo-guanidine IHC image in a CTN.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
So far the benign CTN constitutes a molecular black box, with no causal explanation for their molecular etiology. This is particularly relevant, because benign CTNs represent a frequent scintiscan finding in patients with nodular thyroid disease and need to be distinguished from malignant CTNs (2, 33).

Although most studies have focused on molecular aspects of thyroid tumors with distinct morphological features, e.g. papillary thyroid cancer or follicular adenoma and carcinoma, we here have chosen a phenotypic approach to the entity benign CTN. Thus, we have investigated solitary CTNs (follicular adenoma and adenomatous nodules) in comparison with normal ST of the same patient. We have applied proteomics as a global screening tool, because it permits the identification of protein patterns, which may be specific for the pathological situation under investigation and thus may allow direct deductions as to deregulated in vivo protein function.

In this work, we demonstrate for the first time that 2D-GE in combination with MS was successful to unravel novel pathological features of CTNs. Based on these investigations, the key aspects of CTNs can be summarized as follows (Fig. 9Go).


Figure 9
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  		FIG. 9. Functional model of the pathology of a CTN. I, Iodide enters the follicular cell at the basolateral membrane via active transport by NIS. In most CTNs, NIS is dislocated in the cytoplasm resulting in intrathyroidal iodine deficiency of CTNs. II, Iodination of Tg and coupling of the iodotyrosines to form T3 and T4 at the apical membrane. This H2O2-dependent reaction is catalyzed by TPO, and H2O2 is supplied by the ThOX1/2 enzyme system. ThOX1/2 and TPO are up-regulated in CTNs. The antioxidant proteins PDRX-2/6/5, DJ-1, and GST-{pi} are induced to scavenge futile H2O2 produced by the ThOX system in CTNs. III, Tg is stored in the follicle lumen in a highly condensed and covalently cross-linked form. A failure in Tg iodination will lead to an accumulation of T4-poor, insufficiently iodinated Tg. We detected a complete absence of T4-Tg in all analyzed CTNs by IHC as well as the absence of the 15-kDa hormonogenic band on SDS-PAGE. IV, CB mediates the proteolysis of Tg at the apical membrane, thereby liberating T4. Lack of iodinated Tg could lead to an increased expression of CB. Up-regulated CB was detected in CTNs compared with STs. V, Tg is internalized from the lumen and is hydrolyzed by, e.g. CB, thereby releasing T3 and T4. VI, Because of the increased endocytosis of Tg from the colloid as a consequence of intrathyroidal iodine deficiency, Tg proteolysis and degradation is increased. Increased formation of several Tg components was detected exclusively in CTNs.

 
First, CTNs are characterized by intrathyroidal iodide deficiency, prominently as a consequence of a failure in membrane targeting of NIS or reduced NIS expression, which has previously been reported by several other investigators and was confirmed in this study. However, no structural NIS defects have been detected in CTNs until now (9, 10). Another possible mechanism contributing to hypofunctionality of a CTN could be deficient iodine organification. In fact, monoallelic loss of the TPO gene has been described by Krohn and Paschke (11) in benign CTNs compared with normal thyroid tissue, but no somatic TPO mutations have been identified in CTNs.

Second, the intrathyroidal iodide deficiency is not accompanied by down-regulation of the thyroid hormone synthesis apparatus but conversely by the up-regulation of TPO and ThOX1/2 in CTNs compared with normal thyroid tissue. This is particularly remarkable, because it suggests a compensatory mechanism. However, and third, apparently the up-regulation of TPO and ThOX1/2 cannot compensate for iodide deficiency resulting in decreased iodothyronine formation on the Tg molecule. This is evidenced by the absence of T4-Tg staining in CTN as determined by IHC and the absence of an essential hormonogenic 18-kDa peptide of Tg in CTNs (25).

Fourth, additional results gained in this study indicate an increased Tg turnover in CTNs, which most likely might be a consequence of deficient thyroid hormone production. This is suggested by 1) up-regulation of CB, which is involved in apical Tg proteolysis resulting in thyroxine release, 2) the presence of Tg fragments in CTNs, and 3) the induction of several molecular chaperones, which might be induced by a higher Tg production and/or Tg proteolysis occurring with increased Tg turnover. Clearly, there is no evidence for causal structural Tg abnormalities in CTNs.

Possibly, the most striking aspect of our study is the finding of up-regulation of the H2O2 generator system, which suggests the up-regulation of the protein kinase C (PKC) pathway in CTNs. Besides the protein kinase A pathway, the PKC pathway regulates the expression of functional proteins such as NIS and TPO (32, 34). This is in good agreement with recent microarray analysis by Eszlinger et al. (35) showing up-regulation of PKC-related genes in benign CTNs.

Moreover, the up-regulation of the H2O2 generator system is accompanied by the up-regulation of the detoxifying system in CTNs, although this may not be fully counterbalanced. This is particularly suggested by increased formation of 8-oxo-guanidine DNA adducts in CTNs compared with STs. There are several ways in which a cell can deal with 8-oxo-guanidine formation, i.e. through repair mechanisms, by entering apoptosis, or by necrosis (36, 37, 38). In addition, radicals can cause DNA mutations, which if unrepaired and not lethal, will be propagated in a proliferating, clonal lesion. In addition, CTNs exhibit up-regulation of the APP in CTNs, for which a proliferative effect on thyroid epithelial cells has been demonstrated (22). Together with the increased Ki67 expression in CTNs (39) and up-regulation of several cell cycle components, identified in a recent microarray analysis of CTNs (10, 35), this strongly suggests an inherent growth potential in CTNs. Thus, on the basis of the simultaneous occurrence of increased of 8-oxo-guanidine DNA adducts and an increased proliferation rate in CTNs, it is tempting to speculate that these factors could propel mutagenesis and thyroid cell dedifferentiation in CTNs.

Although we were able to unravel several novel pathogenic features of benign CTNs, other important aspects, e.g. the underlying genetic defect(s), remain unresolved. Furthermore, it is still unclear whether in the context of multistep carcinogenesis a CTN represents a true dedifferentiated lesion or is but a benign thyroid lesion with loss of iodine uptake that harbors no additional risk for malignancy. Even though clinical practice shows that only a small minority of CTNs are cancerous, based on the findings presented here, we are more inclined toward the first scenario. Further resolution of these issues will be elementary for both follow-up and treatment recommendations in patients with benign CTNs.


   Acknowledgments
 
We thank Monika Gutknecht for excellent technical help and support and are grateful to Prof. J. Kratzsch, Institute for Clinical Biochemistry Leipzig, and BRAHMS AG for the thyroglobulin measurement. We thank the following for the kind provision of antibodies: Dr. Costagliola and Dr. Miot (IRBN Brussels), Dr. Ruf (University Marseille), Dr. Knoops (Universite Catholique de Louvain), and Prof. de Vijlder (University of Amsterdam). We thank Prof. Lamesch, Department of Surgery Leipzig; Dr. Mühl, Department of Surgery Wurzen; and Prof. Tannapfel, Institute of Pathology, for the supply of thyroid tissue samples.


   Footnotes
 
This study was supported by the Deutsche Forschungsgemeinschaft (Emmy Noether Nachwuchsgruppe to D.F., Fu356/1-2).

Disclosure Summary: The authors have nothing to disclose.

First Published Online December 28, 2006

Abbreviations: APP, Amyloid precursor protein; APP-BP, amyloid-ß- A4 binding protein; CB, cathepsin B; CTN, cold thyroid nodule; 2D-GE, two-dimensional gel electrophoresis; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GST, glutathione S-transferase; HSP, heat-shock protein; IHC, immunohistochemistry; IPG, immobilized pH gradient; MS, mass spectrometry; MW, molecular weight; NIS, sodium iodide symporter; PDI, protein disulfide isomerase; PDRX, peroxiredoxin; pI, isoelectric point; PKC, protein kinase C; ST, surrounding tissue; Tg, thyroglobulin; ThOX, thyroid oxidase; TPO, thyroid peroxidase; RuBPS, ruthenium II Tris bathophenanthroline disulfonate.

Received June 6, 2006.

Accepted for publication December 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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