Selenoproteins of the Thyroid Gland: Expression, Localization and Possible Function of Glutathione Peroxidase 3

Home , GPX2 (gene), GPX3, Thyroid peroxidase

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Biol. Chem., Vol. 388, pp. 1053–1059, October 2007 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2007.122

Selenoproteins of the thyroid gland: expression, localization and possible function of glutathione peroxidase 3

Cornelia Schmutzler1,*, Birgit Mentrup1,a, Lutz Introduction: the thyroid gland, thyroid Schomburg1, Cuong Hoang-Vu2, Volker Herzog3 hormones and selenium and Josef Ko¨ hrle1 The main physiological function of the thyroid gland is 1 Institut fu¨ r Experimentelle Endokrinologie, Charite´– the synthesis of thyroid hormones (TH), for which it is the Universita¨ tsmedizin Berlin, Charite´ platz 1, exclusive source in the vertebrate body. THs are key D-10117 Berlin, Germany regulators of development, growth, and differentiation, 2 Experimentelle und Chirurgische Onkologie, Martin- particularly of the nervous system, as well as many phys- Luther-Universita¨ t Halle/Wittenberg, Magdeburger Str. iological processes in the adult, such as body tempera- 18, D-06097 Halle/Saale, Germany ture, heart activity and energy metabolism. THs are 3 Institut fu¨ r Zellbiologie, Rheinische Friedrich-Wilhelms- synthesized by the epithelial cells of the thyroid gland, Universita¨ t, Ulrich-Haberland-Str. 61a, D-53121 Bonn, the thyrocytes (Taurog, 2005). These cells line the lumen Germany of the thyroid follicles, which contain the so-called col- * Corresponding author loid, mainly consisting of thyroglobulin (Tg) and small e-mail: [email protected] amounts of other proteins. The three crucial steps of TH synthesis are all catalyzed by a single enzyme, the heme protein thyroid peroxidase (TPO) and comprise: (1) the oxidation of iodide; (2) the iodination of ‘hormonogenic’ tyrosine residues of Tg; and (3) the oxidative coupling of two iodinated tyrosine residues to give Tg-bound TH thy- Abstract roxine (T4) and triiodo-thyronine (T3). Tg is secreted in dimeric form (2=330 kDa, 19 S) towards the apical lumen The thyroid gland has an exceptionally high selenium of the thyroidal follicle. After iodination and coupling, the content, even during selenium deficiency. At least 11 colloidal Tg with the bound TH may be proteolyzed either selenoproteins are expressed, which may be involved in in the follicular space or within lysosomes after micro- the protection of the gland against the high amounts of pinocytotic uptake. This eventually leads to TH release into the circulation. Alternatively, Tg forms highly poly- H2O2 produced during thyroid hormone biosynthesis. As determined here by in situ hybridization and Northern merized ‘storage Tg’ deposited in the colloid lumen (see blotting experiments, glutathione peroxidases (GPx) 1 below). Thus, Tg is another central player in the reaction and 4 and selenoprotein P were moderately expressed, sequence of TH biosynthesis. occurring selectively in the follicular cells and in leuko- An essential co-substrate for TPO in this pathway is H O , which provides oxidative equivalents for TPO and cytes of germinal follicles of thyroids affected by Hashi- 2 2 is produced in high amounts by the NADPH-dependent moto’s thyroiditis. Selenoprotein 15 was only marginally flavoproteins thyroid oxidase DUOX (dual oxidases) 1 expressed and distributed over all cell types. GPx3 and 2. Thus, as a consequence of its normal physiolog- mRNA was exclusively localized to the thyrocytes, ical activity, the gland is continuously exposed to relevant showed the highest expression levels and was down- concentrations of H O (in addition to the ‘normal’ share regulated in 5 of 6 thyroid cancer samples as compared 2 2 of a cell, which is contributed by mitochondrial metabo- to matched normal controls. GPx3 could be extracted lism) and to H O -derived reactive oxygen species (ROS) from thyroidal colloid by incubation with 0.5% sodium 2 2 probably arising as by-products of these processes. dodecyl sulfate indicating that this enzyme is (i) secreted Although TH synthesis is compartmentalized to the into the follicular lumen and (ii) loosely attached to the lumen of the follicles, and both the DUOX enzymes and colloidal thyroglobulin. These findings are consistent with TPO are localized to the apical membrane of the thyro- a role of selenoproteins in the protection of the thyroid cyte, H O can freely diffuse into the cytoplasm and from possible damage by H O . Particularly, GPx3 might 2 2 2 2 nucleus, where it may lead to aberrant oxidation and iodi- use excess H2O2 and catalyze the polymerization of thyroglobulin to the highly cross-linked storage form nation of proteins and lipids, trigger apoptosis and induce DNA damage. However, the H O produced is present in the colloid. 2 2 consumed at least in part to form a special ‘storage Tg’. This form consists of highly polymeric Tg cross-linked by Keywords: deiodinase; glutathione peroxidase 1; intra- and intermolecular disulfide bonds and is depos- glutathione peroxidase 3; glutathione peroxidase 4; ited in the follicular lumen in protein concentrations of up selenoprotein 15; selenoprotein P; thyroglobulin. to 590 mg/ml as insoluble protein globules (Berndorfer et al., 1996). In the current model, it is assumed that the aPresent address: Max-Planck-Institut fu¨ r Molekulare Genetik, formation of these covalent bonds is also catalyzed by Fabeckstr. 60-62, D-14195 Berlin, Germany. TPO.

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1054 C. Schmutzler et al.

The thyroid gland, together with the brain and several stimulation by TSH triggering of compensatory growth of other endocrine tissues, is among the organs with the the thyroid as well as – inadequately – increased H2O2 highest selenium (Se) content in vertebrates (Dickson and production for TH synthesis. However, the latter cannot

Tomlinson, 1967; Behne et al., 1988). Furthermore, under be counter-regulated due to inadequate function of H2O2- conditions of Se deficiency, the gland retains or even scavenging selenoenzymes, resulting in a ‘vicious circle’ accumulates Se, as shown in rats exposed to nutritional of stimulation and increased tissue damage followed by Se shortage (Behne et al., 1988; Bermano et al., 1995). thyroid necrosis and, finally, fibrosis. Other factors, such These findings have been impressively verified in a as deficiency in further trace elements, may also contrib- genetic model for Se deficiency, the selenoprotein P ute to pathogenesis. In rats, Se deficiency aggravates the (SePP) knockout mouse, which lacks the plasma protein susceptibility to inflammation and necrosis of the thyroid SePP, the main Se distributor in the vertebrate organism. gland caused by iodide overload in iodine-deficient thy- SePP knockout mice display decreased serum Se levels, roid glands; TGFb seems to play a prominent role in this with manifest growth defects and neurological abnor- process (reviewed by Ko¨ hrle et al., 2005). However, con- malities indicating Se deficiency in the central nervous tradictory data have also been published (Colzani et al., system (Hill et al., 2003; Schomburg et al., 2003). Sur- 1999). prisingly, however, thyroid Se concentration, thyroid Thyroid carcinoma, although it comprises only 1% of glutathione peroxidase (GPx) activity, thyroid gland mor- all cancers diagnosed, is the most frequent malignant phology, and serum levels of T4, T3 and TSH were within disease of the endocrine system, and the anaplastic var- normal range. Thus, the thyroid gland occupies a partic- iant is one of the most deadly cancers, with a mean sur- ularly high rank in the Se hierarchy that regulates the pri- vival rate of only a few months after diagnosis (Pasieka, ority according to which various tissues obtain their share 2003). The Norwegian JANUS control study demonstrat- of a limited pool of Se. Accordingly, at least 11 seleno- ed an elevated risk of developing thyroid carcinoma proteins occur in the thyroid gland (Behne et al., 1988), associated with prediagnostically low plasma Se concen- including 59-deiodinases type I and type II (59DI, 59DII), trations (Glattre et al., 1989). Another study examined glutathione peroxidases GPx1, GPx3 and GPx4, thiore- concentrations of minerals in the blood and thyroid tissue doxin reductase (TrxR), the Se transport protein SePP of several patients with thyroid diseases and found that and selenoprotein 15 (SeP15), where they may catalyze patients with thyroid cancer had the lowest mean Se lev- redox reactions involved in various physiological pro- els (Kucharzewski et al., 2003). cesses such as the scavenging of ROS, TH metabolism Accordingly, in thyroid cancer, the expression of vari- and others (Ko¨ hrle, 2005; Ko¨ hrle et al., 2005). ous selenoproteins is dysregulated. 59DI expression and The observations summarized above indicate that an enzyme activity are decreased or absent in thyroid car- adequate Se supply is required for proper thyroid func- cinoma tissues and cell lines (Ko¨ hrle, 1997). DNA array tion. This conclusion is supported by additional data link- data indicate that 59DI is down-regulated in follicular thy- ing several pathological conditions of the thyroid gland roid adenoma compared to follicular thyroid carcinoma, to Se status. For example, patients with autoimmune thy- and the same is true for SePP (Barden et al., 2003). Sele- roiditis (AITD) seem to benefit from Se supplementation. nium binding protein 1 (SELENBP1; Chang et al., 1997) AITD is one of the most frequent organ-specific autoim- is not a classical selenoprotein in the sense that it does mune diseases and affects approximately 2% of the pop- not contain selenocysteine, but rather harbors Se atoms ulation, with a five- to ten-fold excess in women over bound in a non-covalent form. SELENBP1 was highly men (Vanderpump et al., 1995). In a study performed in expressed in normal thyroid gland, as shown by immuno- an area with moderate Se deficiency, supplementation cytochemistry (Kim et al., 2006), but down-regulated in with 200 mg Se/day in the form of sodium selenite for papillary thyroid carcinoma, as shown by proteomic anal- 3 months significantly reduced TPO autoantibody (TPO- ysis (Brown et al., 2006). A DNA array approach showed Ab) titers and improved ultrasound echogenicity of the that SELENBP1 mRNA was underexpressed in follicular thyroid gland, as well as the subjective wellbeing of the thyroid carcinoma compared to follicular thyroid adeno- patients (Ga¨ rtner et al., 2002). A follow-up crossover ma (Chevillard et al., 2004). Finally, studies using serial study for a further 6 months was performed (Ga¨ rtner and analysis of gene expression (SAGE) suggested that GPx3 Gasnier, 2003). In the groups that continued taking Se or is down-regulated in thyroid carcinoma (Takano et al., started Se supplementation after having received place- 2000; Hasegawa et al., 2002). Our own data obtained by bo for the first 3 months, TPO-Ab concentrations further in situ hybridization also indicated reduced GPx3 mRNA significantly decreased. In the group that stopped taking expression in Hu¨ rthle cell carcinoma (Menth et al., 2005). Se, TPO-Ab concentrations rebounded towards presup- In contrast to the selenoproteins mentioned above, TrxR plementation concentrations. Two other studies, one also was overexpressed in thyroid cancer, as well as in can- performed in a region with mild Se deficiency (Turker et cers of many other tissues (Lincoln et al., 2003), and al., 2006) and the other conducted in a Se-adequate SAGE analysis showed overexpression of SeP15 mRNA region (Duntas et al., 2003), supported these results. in papillary thyroid carcinoma (Ris-Stalpers et al., 2001). Se shortage is particularly deleterious in combination Which are the roles of Se and selenoproteins in sup- with inadequate iodine supply and possibly also with porting proper thyroid function and protecting the thyroid exposure to goitrogens such as thiocyanates (reviewed from adverse effects? Given the high concentration of by Ko¨ hrle et al., 2005). This is observed in some regions H2O2 in the thyroid during hormone synthesis, it is con- of Africa and may be the cause for myxedematous cre- ceivable that effective protection against H2O2-derived tinism. Here, thyroid insufficiency leads to increased ROS is required. So far, it has been proposed that two

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Selenoproteins of the thyroid gland 1055

selenoenzymes provide for detoxification of H2O2 in the thyrocytes. Ekholm and Bjo¨ rkman (1997) showed that GPx-1 is able to prevent aberrant intracellular iodination by metabolizing H2O2. As for GPx3, Howie et al. (1995) detected secretion of the enzyme into the conditioned medium of quiescent primary human thyrocytes, sug- gesting that the enzyme may metabolize extracellular

H2O2. In contrast, under stimulation by TSH, which increases H2O2 production and TH biosynthesis, secretion of GPx3 is reduced and the enzyme remains within the cell. In vivo, this may guarantee that the TPO co- substrate H2O2 is available for hormone synthesis in the follicular lumen, while GPx3 participates in intracellular detoxification of H2O2, together with GPx1 and catalases. This model, however, suggests a function exceeding that of a mere ROS-scavenging protein and indicates a role for GPx3 in the regulation of TH production.

In addition to detoxification of H2O2 and regulation of TH synthesis, GPx3 may fulfill a third function: by con- suming H2O2 it might actively take part in the polymerization process leading to the formation of cross-linked storage Tg, an enzymatic function that is, as stated above, currently assigned to TPO. It is also conceivable that GPx3, by analogy to the ‘moonlighting’ of GPx4 during sperm formation (Ursini et al., 1999), polymerizes itself and/or is cross-linked to the Tg polymer. As a pre- requisite to clarification of this assumption, we studied the expression and localization of GPx3 in the thyroid gland.

Results

We used in situ hybridization to study the localization of various selenoproteins within the thyroid gland using goiter, AITD or thyroid tumor samples (Figure 1). The Figure 1 Expression of selenoproteins in the thyroid gland strongest hybridization signals were obtained from GPx3 determined by in situ hybridization. mRNA, allowing detection after only approximately cDNA fragments coding for various selenoproteins were labeled with w35Sx-UTP and hybridized to 12-mm cryosections of human 10 days of exposure of the tissue slides. GPx3 was con- thyroid tissue. Small insets show the hybridization reactions with fined exclusively to the thyrocytes (Figure 1A–C). In thy- the corresponding sense probes, which were invariably negative. roid carcinomas, the follicular structure of the normal (A) GPx3 probe hybridized to a colloid goiter sample; (B) GPx3/ thyroid was disrupted and GPx3 signals were evenly dis- follicular adenoma; (C) GPx3/Hashimoto’s thyroiditis; (D) GPx3/ persed over the whole sample areas or parts of it (Figure thyroid carcinoma; (E) GPx4/follicular adenoma; (F) GPx4/Hashi- 1D), without any of the thyroid-specific differential distri- moto’s thyroiditis; (G) GPx1/follicular adenoma; (H) GPx1/Hash- imoto’s thyroiditis; (I) SePP/goiter; (J) SePP/Hashimoto’s thyroid- bution patterns observed, e.g., in goiter (Figure 1A). itis; and (K) SeP15/colloid goiter. GPx3 expression is confined Detection of GPx4 and GPx1 mRNAs required exposure to thyrocytes, whereas other selenoproteins also occur in addi- of approximately 100 days. GPx4 mRNA expression was tional cell types. also strongest in thyrocytes; however, in samples from Hashimoto’s thyroids, leukocytes contained in germinal Results obtained from Northern blot analysis using a follicles showed detectable hybridization signals. GPx1 multiple tissue mRNA expression blot confirmed these mRNA gave the weakest hybridization signals; further- data. As for GPx3, kidney, the organ producing GPx3 for more, in Hashimoto’s samples, they were barely detect- excretion into the serum (Yoshimura et al., 1991), exhib- able in thyrocytes, and were strongest in leukocytes of ited the strongest hybridization signal (100%), with the the germinal follicles. The same pattern was also second strongest signal observed for the thyroid gland observed for SePP mRNA, although in this case the sig- (37%) (Figure 2A); all other tissues showed far lower nals were even weaker than for GPx1. SePP was also expression levels. The highest GPx1 mRNA expression found in single cells interspersed between thyroidal fol- (data not shown) was observed in adrenal gland (100%), licles, which might represent C cells. There were very low in leukocytes (93%) and in thyroid gland (92%). GPx4 levels of SeP15 mRNA expression; signals were homo- (data not shown) was most highly expressed in testis geneously distributed over all cell types and did not show (100%), where this selenoprotein not only has an enzy- any specific expression pattern. matic function, but also serves as a structural protein

1056 C. Schmutzler et al.

Figure 2 Northern blot analysis of selenoprotein expression in Figure 3 Localization of GPx3 to the follicular colloid of the the thyroid gland. thyroid gland. cDNA fragments coding for the selenoproteins GPx3, SePP and Colloidal Tg was prepared in globular form from post mortem SeP15 were labeled with w32PxdCTP and hybridized to dot blot thyroid glands and incubated under denaturing (0.5% SDS) or arrays containing RNAs from various human tissues (A) or from reducing (50 mM DTT) conditions to solubilize the polymeric Tg carcinoma samples together with matched normal tissue (B). and additional proteins. (A) Thyroid globules before complete GPx3 is highly expressed in kidney and thyroid gland, whereas removal of the lining follicular epithelium (left) and after complete other tissues exhibit lower expression levels. SeP15 is more uni- separation of globules from thyrocytes (right). (B) Western blot formly expressed in most tissues (A). In thyroid carcinoma, SePP analysis of the various colloidal fractions. Protein homogenate mRNA is down-regulated in 3 of 6 and GPx3 mRNA in 5 of 6 from human goiter or liver served as control. Whereas the secre- tumor samples compared to their matched normal controls (B). tory protein GPx3 is detected in the colloid, the internal membrane protein 59DI is not. (Ursini et al., 1999), followed by kidney (39%) and thyroid gland (27%). SeP15 was more or less uniformly expressed in most of the tissues (Figure 2A). The liver is colloidal Tg. There was no 59DI signal observed, exclud- the organ that produces SePP for the distribution of Se ing any contamination of the colloid with intracellular throughout the organism and thus shows the highest lev- material. el of SePP mRNA expression (100%). SePP signals in thyroid reached only 14% of the intensity observed for liver. When expression of selenoprotein transcripts in Discussion control tissues was compared with thyroid carcinoma, GPx3 mRNA was down-regulated in 5 of 6 samples and Using Northern blot analysis, we found high expression SePP mRNA in 3 of 6 samples compared to matched levels of mRNAs for GPx3, GPx4 and GPx1 in the thyroid normal thyroid tissue (Figure 2B), whereas there was no gland, whereas SePP and SeP15 mRNAs were present alteration in SeP15 (data not shown). only in comparatively moderate amounts. Indeed, regard- To determine the localization of GPx3 within the thyroid ing any GPx isoenzyme except for gastrointestinal GPx2, gland, colloid globules were prepared and partially puri- the thyroid gland was among the organs exhibiting the fied from post mortem thyroid tissue from healthy sub- strongest expression. This underscores an important role jects (Figure 3A). The globules were first treated with for these enzymes in the antioxidative protection of the

0.5% sodium dodecyl sulfate (SDS); this dissolves gland against the high concentrations of H2O2 produced approximately 26% of the Tg, corresponding to loosely during TH biosynthesis. incorporated or surface-attached material. The globules We detected especially high mRNA concentrations for were then incubated in 50 mM dithiothreitol (DTT), which GPx3 in thyrocytes, the TH-producing cells of the thyroid dissolves 74% of the colloid Tg by disruption of covalent gland. As also reported by another group, expression disulfide cross-links. Any other proteins also contained in was down-regulated in thyroid carcinoma (Hasegawa et the colloid are expected to appear in one of the two frac- al., 2002; Menth et al., 2005), indicating coupling of GPx3 tions, depending on whether they are covalently bound expression to the differentiated status of the thyroid to Tg or not. The supernatants of the respective treat- gland, and probably a special thyroid-specific function of ments were analyzed by Western blotting, along with this enzyme. GPx3 is also produced at high levels and untreated colloid as a control. secreted by proximal kidney tubules, which are assumed As observed in Figure 3B, GPx3 was detectable in the to supply circulating GPx3 into the bloodstream. Our colloid. It was possible to recover all of the GPx3 initially unpublished results (Mentrup and Schmutzler) have dem- present in the colloid globuli in the SDS supernatant. No onstrated a binding site for the transcription factor Pax8 GPx3 was observed in the DTT fraction. This indicates in the upstream regulatory region of the GPx3 gene, that GPx3 is indeed secreted into the follicle lumen. How- which was shown to be functional during transcription of ever, it is only loosely attached, but not cross-linked, to the GPx3 gene in transient transfection assays. Pax8 is

Selenoproteins of the thyroid gland 1057 a paired box-containing, redox-regulated transcription Materials and methods factor that is specific for both kidney and thyroid gland. This transcription factor is indispensable for development In situ hybridization of the thyroid, and in the adult thyroid regulates a number of genes coding for thyroid-specific proteins that perform Thyroid tissue specimens obtained from patients undergoing surgery for thyroid disease were a kind gift from H. Feustel (Mis- essential roles in TH biosynthesis, such as TPO and Tg sionsa¨ rztliche Klinik, Wu¨ rzburg, Germany) and C. Hoang-Vu (Missero et al., 1998). In thyroid cancer, Pax8 is down- (Universita¨ t Halle, Halle/Saale, Germany). The study was regulated (Ros et al., 1999) or may be affected by the approved by the local University Ethics Committee and all t(2;3)(q13;p25) chromosomal translocation also involving patients gave written consent. the PPARg1 gene (Kroll et al., 2000). The presence of the cDNA constructs containing the coding regions for human Pax8 binding site in the human GPx3 promoter is in line GPx1, GPx3, GPx4, SeP15 and SePP were generated by RT- with the high expression levels of the enzyme in the thy- PCR amplification using RNAs isolated from human kidney (GPx) roid gland and its down-regulation in thyroid cancer. and the human hepatocarcinoma cell line HepG2 (SePP) as tem- Howie et al. (1995) presented evidence that GPx3 is plates. The amplified cDNAs were cloned into the pGEM vector apically secreted by human thyrocytes in primary culture (Promega, Mannheim, Germany) and labeled by transcription in w35 x under hormonal control via the Ca2q/IP second messen- the presence of S -UTP (Amersham, Freiburg, Germany) using 3 SP6 and T7 RNA polymerase (Life Technologies, Eggenstein, ger pathway, whereby TSH inhibits GPx3 secretion. They Germany) to give antisense probes and sense controls. Contam- proposed that GPx3 might be involved in control of the inating plasmid DNA was then removed by DNaseI digestion. availability of H2O2 for TH biosynthesis: if H2O2 produc- Cryosections (12 mm) of human thyroid tissue samples were tion triggered by TSH is actively proceeding, GPx3 might used for hybridization. These were incubated with labeled RNAs be retained within the cells to enable efficient iodine oxi- (specific activity approx. 180 MBq/mmol) at an activity of dation, tyrosine iodination and coupling and, at the same 50 000 cpm/ml in hybridization buffer (600 mM NaCl, 10 mM Tris- time, act as an intracellular H2O2 scavenger, together with HCl, pH 7.5, 1 mM EDTA, 0.05% tRNA, 1= Denhardt’s solution, GPx1 and the catalases (Ekholm and Bjo¨ rkman, 1997). 10% dextran sulfate, 100 mg/ml salmon sperm DNA, 50% for- We propose the hypothesis that GPx3 is also involved mamide, 2% DTT) overnight at 588C in a humid chamber. After in Tg polymerization in the follicular colloid, where Tg is incubation, sections were washed, with the most stringent step deposited in a highly polymeric storage form with Tg at 0.2= SSC (30 mM NaCl, 3 mM sodium citrate) and 608C for molecules containing intermolecular cross-links via disul- 1 h, dehydrated with ethanol, dried and dipped into photograph- ic emulsion (Amersham). After an adequate interval (ca. 10 days fide bridges. These might involve the three so-called for GPx3 or 3 months for GPx1, GPx4 and SePP) of exposure CXXR thioredoxin motifs in the mid-region of Tg (Klein et at 48C, samples were developed, fixed, counter-stained with al., 2000). (Excessive) H2O2 that is not consumed during hemalaun (Merck, Darmstadt, Germany) and mounted with iodination and coupling of Tg tyrosine residues is instead Entellan (Merck). used for oxidation of SH groups of Tg. The current model postulates that TPO catalyzes this process. However, Preparation of colloid globules TPO is a membrane protein and is thus fixed within the lumen. Freely diffusible GPx3 might therefore represent Human thyroid tissues, a kind gift of R. Bu¨ ttner (Department of a much more adequate resource to exert this function. Pathology, University of Bonn, Germany), were obtained by GPx4 is a unique enzyme that directly reduces lipid autopsy at approximately 12 h post mortem from patients with- hydroperoxides in membranes and has the ability to use out any detectable disease states of the thyroid. Experimenta- protein thiol groups as donor substrates. It is remarkable tion with human thyroid tissue samples was conducted in that testis exhibits the highest specific activity of GPx4 accordance with the ethical principles and guidelines of the Medical Faculty of the University of Bonn as approved by the so far measured in mammalian tissues, being almost two governmental authorities. orders of magnitude higher than that in brain and liver. For preparation of colloid globules, human thyroid tissues During sperm maturation, however, GPx4 changes its were first minced using razor blades, homogenized at room tem- physical characteristics and biological functions, as the perature in phosphate-buffered saline (PBS) using an Ultra Tur- enzyme exists as a soluble peroxidase in spermatids, but rax homogenizer (15 s; position 4.5; IKA, Taquara, Brazil) in the persists in mature spermatozoa as an enzymatically inac- presence of protease inhibitors (1 mM Na-p-tosyl-L-arginimethyl tive, oxidatively cross-linked, insoluble protein. In the ester, 1 pg/ml antipain, 1 pg/ml pepstatin, 4 pg/ml aprotinin, and mid-piece of mature spermatozoa, GPx4 protein repre- 0.5 mM phenylmethylsulfonyl fluoride). The homogenate was fil- sents at least 50% of the capsule material that embeds tered (150-pm Thermapor nylon gauze, Reichelt Chemietechnik, the helix of mitochondria, thus serving as a structural Heidelberg, Germany) and the filtrate was centrifuged at 100 g protein rather than an enzyme in this special situation for 1 min. The sediment was repeatedly resuspended in PBS (Ursini et al., 1999). We hypothesized that GPx3 secreted and sedimented again. This washing procedure was repeated until the supernatant appeared clear. When examined by light to the follicular colloid might become cross-linked in a microscopy, the sediment was highly enriched in colloid glob- similar manner to Tg. However, all GPx3 present in Tg ules and was essentially free of thyroid connective tissue, adher- globules can be extracted by incubation with 0.5% SDS, ing thyrocytes and any other visible contaminants. so that no enzyme is left to be liberated by reductive treatment with 50 mM DTT, indicating that this is not the Extraction of GPx3 from colloid globules and case. This observation demonstrates that GPx3 is not Western blotting covalently linked to Tg, but remains, after secretion into the lumen, a soluble protein only loosely attached to the The colloid globules were incubated in 0.5% (w/v) SDS overnight follicular colloid. at room temperature and then centrifuged for 5 min at 10 000 g.

1058 C. Schmutzler et al.

The supernatant was removed and stored for analysis, and the References sediment was incubated in 50 mM DTT overnight at room temperature and centrifuged again as above. The pellet was dis- carded and the supernatant was stored for analysis. Barden, C.B., Shister, K.W., Zhu, B., Guiter, G., Greenblatt, D.Y., Zeiger, M.A., and Fahey, T.J. III (2003). Classification of follic- For Western blot analysis, thyroid tissue was first powdered ular thyroid tumors by molecular signature: results of gene using a micro dismembrator (Braun, Melsungen, Germany) and profiling. Clin. Cancer Res. 9, 1792–1800. then sonified (20 pulses of 0.6 s at 200 W) in homogenization Behne, D., Hilmert, H., Scheid, S., Gessner, H., and Elger, W. buffer (250 mM glucose, 20 mM HEPES, 1 mM EDTA and 1 mM (1988). Evidence for specific selenium target tissues and new DTT). Protein concentrations were determined using the modi- biologically important selenoproteins. Biochim. Biophys. fied Bradford assay kit and a preparation of human IgG as stan- Acta 966, 12–21. dard, both from Bio-Rad (Munich, Germany). Loading buffer Bermano, G., Nicol, F., Dyer, J.A., Sunde, R.A., Beckett, G.J., w 400 mM Tris-HCl, 4% (w/v) lithium dodecyl sulfate, 30% (v/v) Arthur, J.R., and Hesketh, J.E. (1995). Tissue-specific regu- glycerol, 204 mM mercaptoacetic acid, 0.02% (w/v) bromophe- lation of selenoenzyme gene expression during selenium nol blue, pH 6.8x was added to the samples (50 mg of protein), deficiency in rats. Biochem. J. 311, 425–430. and proteins were separated by SDS polyacrylamide gel elec- Berndorfer, U., Wilms, H., and Herzog, V. (1996). Multimerization trophoresis on gels prepared from 0.8= MDE solution (Biozym, of thyroglobulin (TG) during extracellular storage: isolation of Hessisch Oldendorf, Germany), 375 mM Tris, pH 8.8, 0.1% SDS highly cross-linked Tg from human thyroids. J. Clin. Endo- polyacrylamide in 192 mM glycine, 25 mM Tris, 0.1% SDS, pH crinol. Metab. 81, 1918–1926. 8.8. Proteins were then electroblotted to a stabilized nitrocellu- Brown, L.M., Helmke, S.M., Hunsucker, S.W., Netea-Maier, R.T., lose membrane (Optitran BA-S, Schleicher & Schuell, Dassel, Chiang, S.A., Heinz, D.E., Shroyer, K.R., Duncan, M.W., and Germany) using a Mini Protean unit (Bio-Rad) and stained with Haugen, B.R. (2006). Quantitative and qualitative differences Ponceau S. After blocking w2.5% non-fat milk powder, 2.5% in protein expression between papillary thyroid carcinoma bovine serum albumin (BSA), 2% horse serum, 0.1% Tween 20 and normal thyroid tissue. Mol. Carcinog. 45, 613–626. in PBSx, membranes were incubated with antibody raised Chang, P.W., Tsui, S.K., Liew, C., Lee, C.C., Waye, M.M., and against aa 236–249 at the C-terminus of human GPx3 Fung, K.P. (1997). Isolation, characterization, and chromo- (immunoGlobe, Himmelstadt, Germany). The antiserum was somal mapping of a novel cDNA clone encoding human sele- diluted 1:750 in PBS containing 1% non-fat milk powder, 1% nium binding protein. J. Cell. Biochem. 64, 217–224. BSA, 1% horse serum, 0.1% Tween 20 overnight at 48C. As a Chevillard, S., Ugolin, N., Vielh, P., Ory, K., Levalois, C., Elliott, control, we also used an antiserum directed against human 59DI D., Clayman, G.L., and El-Naggar, A.K. (2004). Gene expres- (antiserum 1068; diluted 1:2000), which was generated using a sion profiling of differentiated thyroid neoplasms: diagnostic peptide derived from aa 236–249 of the protein (Kuiper et al., and clinical implications. Clin. Cancer. Res. 10, 6586–6597. 2003). Membranes were washed four times for 10 min with Colzani, R.M., Alex, S., Fang, S.L., Stone, S., and Braverman, L.E. (1999). Effects of iodine repletion on thyroid morphology 10 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 2 mM EDTA, in iodine and/or selenium deficient rat term fetuses, pups and 1% non-fat dry milk, 1% BSA, 1% horse serum and then incu- mothers. Biochimie 81, 485–491. bated with secondary anti-rabbit antibody coupled to horserad- Dickson, R.C. and Tomlinson, R.H. (1967). Selenium in blood and ish peroxidase (DAKO, Hamburg, Germany; Sigma-Aldrich human tissues. Clin. Chim. Acta 16, 311–321. Chemie; Promega GmbH, Mannheim, Germany) diluted 1:10 000 Duntas, L.H., Mantzou, E., and Koutras, D.A. (2003). Effects of in PBS containing 1% non-fat milk powder, 1% BSA, 1% horse a six month treatment with selenomethionine in patients with serum, 0.1% Tween 20. After another four washes for 10 min autoimmune thyroiditis. Eur. J. Endocrinol. 148, 389–393. (10 mM Tris-HCl, 1 M NaCl, 0.1% Triton X-100, 2 mM EDTA, 1% Ekholm, R. and Bjo¨ rkman, U. (1997). Glutathione peroxidase non-fat dry milk, 1% BSA, 1% horse serum), GPx3 and 59DI degrades intracellular hydrogen peroxide and thereby inhibits were detected using an ECL system (Amersham Buchler, intracellular protein iodination in thyroid epithelium. Endocri- Braunschweig, Germany) as recommended by the manufacturer. nology 138, 2871–2878. Ga¨ rtner, R. and Gasnier, B.C. (2003). Selenium in the treatment Northern blotting of autoimmune thyroiditis. Biofactors 19, 165–170. Ga¨ rtner, R., Gasnier, B.C., Dietrich, J.W., Krebs, B., and Angst- Northern blot analysis was performed using a Human Multiple wurm, M.W. (2002). Selenium supplementation in patients Tissue Expression Array 2 (MTE) 2 and a Cancer Profiling with autoimmune thyroiditis decreases thyroid peroxidase assay purchased from BD Biosciences Clontech (Heidelberg, antibodies concentrations. J. Clin. Endocrinol. Metab. 87, Germany). The array was hybridized to wa32PxdCTP-labeled sele- 1687–1691. noprotein cDNA fragments according to the manufacturer’s pro- Glattre, E., Thomassen, Y., Thoresen, S.O., Haldorsen, T., Lund- tocol. Blots were evaluated using the Cyclone storage phosphor Larsen P.G., Theodorsen, L., and Aaseth, J. (1989). Prediag- system (Packard BioScience, Dreieich, Germany). Signal inten- nostic serum selenium in a case-control study of thyroid can- sities were calculated as digital light units per unit area (DLU/ cer. Int. J. Epidemiol. 18, 45–49. mm2), defined as the specific signal intensity of the hybridizing Hasegawa, Y., Takano, T., Miyauchi, A., Matsuzuka, F., Yoshida, region in mm2 divided by an equally sized area next to the spot H., Kuma, K., and Amino, N. (2002). Decreased expression after subtraction of the background signals. of glutathione peroxidase mRNA in thyroid anaplastic carcinoma. Cancer Lett. 182, 69–74. Hill, K.E., Zhou, J., McMahan, W.J., Motley, A.K., Atkins, J.F., Gesteland, R.F., and Burk, R.F. (2003). Deletion of selenopro- Acknowledgments tein P alters distribution of selenium in the mouse. J. Biol. Chem. 278, 13640–13646. We would like to thank G.G.J.M. Kuiper and T.J. Visser for kindly Howie, A.F., Walker, S.W., Akesson, B., Arthur, J.R., and Beckett, providing the 59DI antiserum, H. Feustel (Wu¨ rzburg) for samples G.J. (1995). Thyroidal extracellular glutathione peroxidase: a of human pathological thyroid tissues and R. Bu¨ ttner (Bonn) potential regulator of thyroid-hormone synthesis. Biochem. for normal thyroid tissue. The excellent technical support of J. 308, 713–717. Mrs. S. Zeck, Mrs. M. Topp and Mrs. B. Bockmu¨ hl (Bonn) was Kim, H., Kang, H.J., You, K.T., Kim, S.H., Lee, K.Y., Kim, T.I., instrumental in this study. This work was supported by Deutsche Kim, C., Song, S.Y., Kim, H.J., Lee, C., and Kim, H. (2006). Forschungsgemeinschaft, grant Ko¨ 922/8-2/3. 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