Thyroid hormone biosynthesis and release

Molecular and Cellular Endocrinology xxx (2017) 1e10

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Thyroid hormone biosynthesis and release Denise P. Carvalho a, *, Corinne Dupuy b, c, d a

Biophysics Institute of Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Universit
e Paris-Saclay, Orsay, France c UMR 8200 CNRS, Villejuif, France d Institut de Canc
erologie Gustave Roussy, Villejuif, Ile-de-France, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 October 2016 Received in revised form 7 January 2017 Accepted 23 January 2017 Available online xxx

Thyroid hormones (TH) 3,5,30 ,5’- tetraiodothyronine or thyroxine (T4) and 3,5,3’- triiodothyronine (T3) contain iodine atoms as part of their structure, and their synthesis occur in the unique structures called thyroid follicles. Iodide reaches thyroid cells through the bloodstream that supplies the basolateral plasma membrane of thyrocytes, where it is avidly taken up through the sodium/iodide symporter (NIS). Thyrocytes are also specialized in the secretion of the high molecular weight protein thyroglobulin (TG) in the follicular lumen. The iodination of the tyrosyl residues of TG preceeds TH biosynthesis, which depends on the interaction of iodide, TG, hydrogen peroxide (H2O2) and thyroid peroxidase (TPO) at the apical plasma membrane of thyrocytes. Thyroid hormone biosynthesis is under the tonic control of thyrotropin (TSH), while the iodide recycling ability is very important for normal thyroid function. We discuss herein the biochemical aspects of TH biosynthesis and release, highlighting the novel molecules involved in the process. © 2017 Published by Elsevier Ireland Ltd.

Keywords: Thyroglobulin TPO NIS DUOX Pendrin Anoctamin-1 ClC5

 Follicular thyroid cells are specialized in thyroid hormone (TH) biosynthesis.  NIS, pendrin, ClC5 and anoctamin-1 are essential for thyroid iodide availability.  Thyroglobulin iodination depends on thyroperoxidase and DUOX2.  MCT8 is a transporter involved in thyroid hormone release.  TSH, iodide, selenium, thyroglobulin and TH metabolites regulate thyroid function.

1. Introduction Thyroid hormone biosynthesis comprises a series of specific biochemical reactions that are closely related to the histological organization of thyroid tissue. Thyroid follicles, which are considered the functional units of the thyroid, are formed by a monolayer of polarized follicular epithelial cells, the so-called thyrocytes that are organized in a tridimensional ovoid structure surrounding the follicle lumen (Fig. 1A). The interior of the follicle primarily contains

* Corresponding author. E-mail address: [email protected] (D.P. Carvalho).

iodinated thyroglobulin (TG) and is called the “colloid” due to the high content of proteins, which are in close contact with the apical plasma membrane of thyrocytes. The exterior of the follicle is delimited by the basolateral plasma membrane of thyrocytes and is in contact with a large network of blood capillaries where intense exchange with the blood occurs (Fig. 1). In follicular cells, the tight junctions form a strong barrier that impairs the diffusion of transmembrane proteins from the apical domain to the basolateral domain, and vice versa. As a result of this intercellular barrier, it is also believed that the follicular luminal content cannot reach the bloodstream through the intercellular spaces unless the barrier is disrupted, as occurs in some pathophysiological circumstances, such as thyroid inflammation. Thyroid hormones contain iodine atoms as part of their molecular structure. However, the initiation of thyroid hormone biosynthetic pathways depends not only on the specificity of iodine metabolism but also on what occurs outside the cells in the follicular lumen at the outer surface of the apical plasma membranes of thyrocytes. This biosynthesis is accomplished thanks to a set of genes that encode transcription factors whose joint expression is characteristic of and specific to thyroid tissue. Altogether, these transcription factors are fundamental for the expression of thyroid differentiation markers, such as TG and other proteins whose

http://dx.doi.org/10.1016/j.mce.2017.01.038 0303-7207/© 2017 Published by Elsevier Ireland Ltd.

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Fig. 1. Schematic Representation of Thyroid Hormone Biosynthesis and Release. The proteins involved in thyroid hormone biosynthesis and release are represented. NIS: sodium/ iodide symporter; KCNQ1 and KCNE2: Voltage-gated Kþ channels; TSHR: thyrotropin receptor; MCT8: SLC16A2 monocarboxylate transporter 8, thyroid hormone transporter; DUOX2: dual oxidase 2; DUOXA2: maturation factor of dual oxidase 2; TPO: thyroperoxidase; DEHAL: iodotyrosine dehalogenase. A- Tridimensional structure of the thyroid follicles that is surrounded by epithelial follicular thyroid cells. C cells are parafollicular cells that produce calcitonin; B- TPO: thyroperoxidase, he: heme group of TPO, Tyr: tyrosine residues of thyroglobulin; C- iodide oxidation and its incorporation into thyroglobulin (organification) depends on the presence of TPO and hydrogen peroxide (H2O2) produced by DUOX2, DIT: diiodotyrosine, MIT: monoiodotyrosine; D- the oxidative coupling of iodotyrosines, MIT and DIT, depends on the presence of TPO and hydrogen peroxide (H2O2) and lead to the formation of T3 (and mainly T4) that remains bound to the thyroglobulin molecule.

localization at the apical plasma membrane allows the iodination of the tyrosyl residues of TG. Some of the iodotyrosine residues formed in the TG core are then coupled to form thyroxine or 3,5,30 ,5’- tetraiodothyronine (T4), through the assembly of two diiodotyrosines (DITs), or 3,5,3'- triiodothyronine (T3), when one monoiodotyrosine (MIT) is coupled to DIT. There are at least four molecules that must interact at the apical plasma membrane of a thyrocyte for thyroid hormone biosynthesis to occur: iodide, TG, hydrogen peroxide (H2O2) and thyroid peroxidase (TPO). Iodide is absorbed in the gastrointestinal tract and reaches the basolateral plasma membranes of thyrocytes through the bloodstream. Although some other tissues are also able to take up iodide from the circulation, the thyroid gland is the only one that avidly

concentrates iodide and accumulates it for a prolonged period of time, as a result of the histologic features of the thyroid follicles and the ability to organify iodine into tyrosyl residues of TG. The biosynthesis of thyroid hormones occurs at the interface of the apical thyroid cell plasma membrane and the colloid, and TG molecules containing T4 and T3 are stored in the follicle lumen. The secretion of thyroid hormones depends on the reabsorption of iodinated TG, its proteolysis and the subsequent release of T4 and T3 into the blood, which occurs at least partially through transporters located in the basolateral plasma membrane of thyrocytes.

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2. Molecules involved in thyroid hormone biosynthesis 2.1. Iodine availability At the end of the 19th century, Baumann (1896) described that the thyroid gland concentrates iodide from the plasma by a factor of 20e40. In 1996, both the rat and human genes that encode the protein responsible for the transport of iodide into thyrocytes were finally sequenced (Dai et al., 1996; Smanik et al., 1996, 1997). The human NIS (hNIS) gene maps to chromosome 19p13.2-p12 and exhibits an 84% amino acid identity and 93% similarity to the rat NIS (rNIS) gene (Smanik et al., 1997). The rNIS gene is markedly smaller in size (9260 bases) than its human counterpart (22,116 bases), although the sizes of their respective mRNA are similar. NIS is a member of the solute carrier transporter family 5 (SLC5), and its secondary structure model predicts a protein with 13 transmembrane segments (Fig. 1), with the amino terminus facing the extracellular milieu, and the carboxy terminus facing the cytosol at the basolateral plasma membrane of thyrocytes (Levy et al., 1997, 1998). NIS-mediated iodide transport is driven by the transmembrane Naþ gradient that is actively generated by the ouabain-sensitive Naþ/Kþ-ATPase pump present at the basolateral plasma membrane of thyrocytes (Fig. 1). Thus, NIS is a secondary active transporter that couples the inward transport of two Naþ ions to the inward translocation of one iodide ion against its electrochemical gradient. The role of the NIS protein in iodide uptake and thus thyroid hormone biosynthesis was confirmed after the description of mutations that are present in patients with dyshormonogenetic goiters followed by the functional studies of the mutated proteins (De la Vieja et al., 2000; De la Vieja et al., 2005; De la Vieja et al., 2007; Dohan et al., 2003). Moreover, the presence of the voltage-gated Kþ channels KCNQ1 and KCNE2 at the basolateral plasma membrane is also important for iodide uptake (Fig. 1), since the disruption of the kcne2 gene in mice also lead to lower thyroid iodide accumulation, goiter and hypothyroidism (Purtell et al., 2012; Roepke et al., 2009). In thyroid cells, iodide transport is stimulated by thyrotropin (TSH) and inhibited by competitive inhibitors such as thiocyanate (SCN) and perchlorate (ClO 4 ) (Dai et al., 1996; Dohan et al., 2001, 2003; Eskandari et al., 1997; Smanik et al., 1996). NIS expression has also been detected in extrathyroid tissues, such as lactating mammary glands, gastric mucosa, salivary and lacrimal glands, choroid plexus, skin, placenta and thymus (Carrasco, 2013). TSH acts in the thyrocytes via a G-protein-coupled receptor (TSHR) that is present in the basolateral plasma membrane of thyrocytes (Fig. 1). TSH stimulates NIS gene expression and increases iodide uptake by thyrocytes via the activation of adenylate cyclase through Gs alpha protein (Kaminsky et al., 1994; Riedel et al., 2001), while it decreases NIS expression through the PI3K pathway that is activated by Gs beta/gamma subunits (Zaballos et al., 2008). NIS gene transcription depends on the presence in the thyroid of a set of transcription factors that includes the paired-domain protein PAX8 and the homeodomain protein NKX2-1 that interact in the NIS promoter region (Ohno et al., 1999). TSH stimulates not only NIS gene transcription and de novo protein synthesis, but it also plays a possible role in the post-transcriptional regulation of NIS (Mendive et al., 2001). NIS protein is present in at least two distinct subcellular compartments: the plasma membrane and intracellular vesicles, the latter being a pool of NIS proteins that can be rapidly mobilized by TSH and other still poorly defined mechanisms (Dohan et al., 2001; Kaminsky et al., 1994; Riedel et al., 2001; Tonacchera et al., 2002).

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The Wolff-Chaikoff effect corresponds to the reduction of iodide transport and its organification into proteins when thyroid cells are exposed to high concentrations of iodine (Price and Sherwin, 1986; Uyttersprot et al., 1997; Wolff and Chaikoff, 1948). This autoregulation of the thyroid function by iodine is transient once thyroid cells are able to escape from the effects of iodine after some days of exposure (Braverman and Ingbar, 1963). Eng et al. (1999) proposed that significantly decreased levels of both NIS mRNA and protein at 1 and 6 days after chronic excess of iodine ingestion by rats could be involved in the mechanism leading to the escape from the Wolff-Chaikoff effect. Later, TPO organification activity was reported to be important for such an inhibition to occur. Indeed, the same treatment is unable to reduce radioiodide transport when TPO activity is concomitantly inhibited by MMI (Ferreira et al., 2005). These results support the idea that organified iodine, and not iodide itself, is responsible for the inhibition of the basolateral transport of I, as previously suggested (Grollman et al., 1986). In addition to TSH and iodine, other factors can influence NIS expression, such as insulin, insulin-like growth factor, transforming growth factor (TGF)-b1, tumor necrosis factor (TNF) a, interferon g, interleukin (IL)-1a, IL-1b, IL-6, and 3-Iodothyronamine (3-T1AM) (Costamagna et al., 2004; Morand et al., 2003; Morgan et al., 2016; Riesco-Eizaguirre et al., 2009; Schanze et al., 2016; SchummDraeger, 2001). In the last decade, new intracellular pathways have been implicated in NIS regulation, and novel insights regarding the regulation of NIS gene expression through epigenetic mechanisms, such as the methylation of the CpG island in the NIS gene promoter region, have raised several hypotheses regarding the loss of NIS ~o expression in hypofunctioning thyroid nodules and cancer (Galra et al., 2013, 2014). Moreover, the activation of PI3K (Zaballos et al., 2008) and mTOR (de Souza et al., 2010) down regulate NIS expression. The activation of AMPK also leads to a decreased NIS protein content (Andrade et al., 2011), in part due to its lysosomal degradation that is a process linked to autophagy (Cazarin et al., 2014). Interestingly, TSH stimulates not only the cAMP/PKA pathway through Gs alpha subunit activation but also the PI3K pathway through Gs beta/gamma activation (Zaballos et al., 2008), whereas it decreases AMPK phosphorylation and activation (Andrade et al., 2011). More recently, the role of reactive oxygen species (ROS) on NIS down regulation after iodide overload has been described by different groups (Arriagada et al., 2015; Leoni et al., 2011; Serrano-Nascimento et al., 2014), and NOX4 was demonstrated to be an important source of ROS involved in NIS down regulation in cancer cells (Azouzi et al., 2016). Interestingly, TSH and selenium have also been shown to cooperate and upregulate NIS expression probably through the increase of Pax8 binding activity on NIS promoter, which depends on its redox state controlled by thioredoxin/thioredoxin reductase-1 (Txn/TxnRd1) reduction of apurinic/apyrimidinic endonuclease 1 (Ape1) (Leoni et al., 2016). It is noteworthy that the presence of functional NIS proteins in the basolateral plasma membrane of thyroid cells is tightly regulated through different mechanisms, as discussed above. After its influx into thyrocytes, iodide is then translocated from the cytoplasm across the apical plasma membrane towards the follicular lumen in a process called “iodide efflux”, which can be mediated by more than one apical channel. Pendrin was the first protein implicated in apical iodide efflux (a Cl/I transporter; Scott et al., 1999; Gillam et al., 2004). Subsequently, the chloride channel ClC5 was also demonstrated to play a role in iodide efflux. This was suggested by the fact that ClC5 knock out animals develop goiters (van den Hove et al., 2006). More recently, another apical transporter involved in iodide efflux was identified to be anoctamin-1

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(Iosco et al., 2014; Twyffels et al., 2014). In summary, the availability of iodide in the follicular lumen depends on its entrance into the thyrocyte through the basolateral plasma membrane, which is mediated by NIS, and on its efflux through the apical plasma membrane that depends on pendrin, ClC5 and anoctamin-1. When iodine organification is impaired, iodide efflux occurs through the basolateral plasma membrane by still undefined mechanisms. Once in the follicle lumen, iodide is incorporated into TG molecules through biochemical reactions that occur outside the thyrocytes in the vicinity of their apical plasma membrane facing the colloid (Fig. 1). 2.2. Thyroglobulin TG exerts two main functions in iodine metabolism: i) it serves as a template for thyroid hormones biosynthesis, ii) and it provides for the intrathyroidal storage of iodine, which is of critical importance for thyroid homeostasis (Dunn and Dunn, 1999, 2001). From a pathophysiological point of view, TG is a major autoantigen in some autoimmune thyroid diseases (Caturegli et al., 1994). Finally, TG gene mutations are the cause of goiters associated with hereditary hypothyroidism (Targovnik, 2013). TG is mainly found as a high molecular weight (660 kDa, TG 19S) glycoprotein homodimer composed of two subunits of 330 kDa each. Monomers are encoded by the TG gene, which is located on chromosome 8q24.2 and comprises 48 exons spread over approximately 270,000 base pairs (270 kb) (Mendive et al., 2001). TG mRNA is the longest major transcript in thyroid cells and produces a protein of 2767 amino acids that includes a signal peptide of 19 residues (Malthiery and Lissitzky, 1987). TG gene expression is not restricted to the thyroid but in thyrocytes it requires the presence of the transcription factors PAX8, NKX2-1 and the forkhead-domain protein FOXE1, and their interaction with the coactivator p300 at its promoter region (Grasberger et al., 2005). The stimulation of thyroid adenylate cyclase by TSH is important to maintain a certain level of TG mRNA expression. TG expression was recently reported to occur also in fibroblasts and fibrocytes expressing the haematopoietic cell antigen CD34 (CD34(þ) fibrocytes), what might be involved in some features of thyroid autoimmunity (Fernando et al., 2014; Smith, 2015). TG undergoes many post-translational modifications prior to its secretion and iodination by the TPO. The N-linked glycosylation of TG occurs in the endoplasmic reticulum and is redesigned in the Golgi apparatus. TG is also phosphorylated on serine/ threonine residues and on carbohydrate units. The importance of some cysteines in both the structure-function relationship and maturation of the TG has been demonstrated in patients harboring TG gene mutations that result in the substitution of cysteine residues of the TG molecule. These patients have congenital or adenomatous goiters caused by the retention of TG in the endoplasmic reticulum (Di Jeso and Arvan, 2016; Hishinuma et al., 1999). Newly synthesized non-iodinated TG proteins are secreted into the follicular lumen via exocytosis through the apical plasma membrane. TSH, via an increase of cellular cAMP, induces a rapid stimulation of exocytosis (Ericson, 1981). The iodination of some aminoacids residues of the newly secreted TG is catalyzed by TPO, which is a transmembrane protein present in the apical plasma membrane. Following oxidation of iodide and iodination that occurs in the colloid-apical plasma membrane interface, TG 19S is modified into more stable proteins by the formation of covalent cross-linkage of TG (disulfide and other inter-chain bridges) resulting in the formation of so-called thyroid globules

(Berndorfer et al., 1996; Herzog et al., 1992). The increase in iodine content per mol of TG seems to be related to the formation of soluble TG multimers, known as TG 27S and TG 37S (Carvalho et al., 1987; Targovnik, 2013).

2.3. Thyroid peroxidase TPO is considered the key enzyme in thyroid hormonogenesis. It catalyzes the oxidation of iodide that is necessary for the iodination of the TG tyrosyl residues (the organification reaction). TPO is also fundamental for the oxidative coupling of hormonogenic iodotyrosine residues into T4 and T3 iodothyronines. Mutations of the TPO gene are responsible for congenital hypothyroid goiters, due to defects in iodine organification (Abramowicz et al., 1992; Carvalho et al., 1994; Ris-Stalpers and Bikker, 2010). Like TG, TPO is a thyroid differentiation marker that also functions as an autoantigen, the socalled “microsomal antigen”, which generates high titers of circulating autoantibodies, particularly in patients with Hashimoto's thyroiditis (Czarnocka et al., 1985). The TPO gene is located on chromosome 2, at 2p25. It comprises 17 exons and produces multiple transcripts via alternative splicing. The major mRNA present in the thyroid contains 17 exons and encodes the protein TPO1, a hemoprotein composed of 919 aminoacids, following the hydrolysis of a signal peptide of 14 amino acids (Kimura et al., 1987). The other transcripts present in normal thyroids undergo the splicing of exons 10, 16, 14 or 8, and encode the proteins TPO2, TPO3 (called “Zanelli”), TPO4 and TPO5, respectively (Ferrand et al., 2003). The TPO gene is almost exclusively expressed in the thyroid and requires at least three transcription factors, PAX8, NKX2-1 and FOXE1, as well as the p300 coactivator (Grasberger et al., 2005; Miccadei et al., 2002). TSH stimulates TPO gene expression via the cAMP pathway (Gerard et al., 1988), and increases TPO levels at the apical plasma membrane by stimulating exocytosis; however, it does not seem to be involved in the direct control of the protein peroxidase activity. Iodide, in its oxidized form as a result of TPO catalysis, seems to exert a negative control on the TPO mRNA level (Morand et al., 2003; Uyttersprot et al., 1997). The TPO1 protein has a transmembrane segment delimiting a short C-terminal intracellular region of approximately 60 amino acids and a long extracellular N-terminal region of 842 amino acids (Fig. 1B). As with TPO1, the TPO3 and TPO4 proteins are enzymatically active, unlike TPO2 and TPO5, each of which lack an essential amino acid at the active site of the enzyme and are rapidly degraded (Ferrand et al., 2003). To date, the physiological role of these different TPO isoproteins is unknown, although they might exert a regulatory role, since a differential expression of thyroperoxidase mRNA splice variants was observed in human thyroid tumors (Le Fourn et al., 2004). Mature TPO proteins are dimers glycosylated at the luminal Nterminal region, which is homologous with that of myeloperoxidase and contains a heme group that is then located outside the thyrocyte (Godlewska et al., 2014). The majority of TPO molecules are detectable within the thyrocyte, and only approximately 20 percent are anchored in the apical plasma membrane. The targeting of TPO to the apical plasma membrane is carried out after the acquisition of the heme group, the glycosylation of asparagine residues in the endoplasmic reticulum, and a redesign of carbohydrate units in the Golgi apparatus. TPO cleavage by an endoprotease seems to be necessary for the processing of the protein (Le Fourn et al., 2005). Covalent binding of the heme group to the apoprotein is autocatalytic, requires H2O2 and promotes its targeting to the apical plasma membrane (Fayadat et al., 1999).

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2.4. H2O2-generating system linked to hormone synthesis or dual NADPH oxidase As every heme peroxidase, TPO cannot oxidize its substrates without first having been oxidized by a molecule of H2O2. The H2O2-generating system linked to hormone synthesis is active at the apical surface of thyrocytes, and only molecules of TPO present at this surface are activated (approximately 20 percent). The first indirect evidence of a biochemical defect in the hydrogen peroxide generating system was described in human cold thyroid nodules, although the biochemical properties of the enzyme were poorly defined at that time (Demeester-Mirkine et al., 1975). It is now wellstablished that the enzyme producing H2O2 linked to hormonogenesis is a calcium-dependent NADPH oxidase (Deme et al., 1985; Virion et al., 1984) that corresponds to dual oxidase 2 (DUOX2) (Ameziane-El-Hassani et al., 2005). Its gene was identified from 1999 to 2000 by different scientific approaches (De Deken et al., 2000; Dupuy et al., 1999). In 2001, the first report of a dyshormonogenetic goiter family with a biochemical defect on the calciumdependent H2O2 generation by thyroid NADPH oxidase was published (Figueiredo et al., 2001). Later, DUOX2 was identified as the enzyme involved in thyroid hormone synthesis based on the fact that mutations of the DUOX2 gene were found to be associated with cases of congenital hypothyroidism due to defects in iodine organification (Grasberger, 2010; Moreno et al., 2002). The DUOX2 gene is located on chromosome 15, in 15q15.3-q21.1. It generates a mRNA of 6532 nucleotides, containing 34 exons of the gene and encoding a protein of 1548 amino acids. Of note, at the same locus, is the DUOX1 gene, which is symmetrically oriented with respect to DUOX2 and is located 17 kb from DUOX2 and extends to telomere. It encodes a protein of 1551 amino acids (DUOX1), 77 percent of which are identical to those of DUOX2. The role of DUOX1 in hormone biosynthesis is currently hypothetical. However, with the growing number of mutations detected in the DUOX2 gene, it is increasingly becoming more difficult to establish a correlation between genotype and phenotype, as initially described (Moreno et al., 2002). Indeed, two partial and transient types of congenital hypothyroidism associated with the complete inactivation of both alleles of DUOX2 have been reported (Hoste et al., 2010; Maruo et al., 2008) suggesting that DUOX1 might compensate, at least partially, for defects in DUOX2. DUOX2, as with its DUOX1 isoenzyme, is a membrane protein with seven transmembrane domains (Fig. 1). The N-terminal ectodomain and the first transmembrane segment are specific for DUOX1/2 and are absent from the five other NADPH oxidases described to date (Ameziane-El-Hassani et al., 2016). Located between the first two transmembrane segments, the large intracellular loop, carrying two EF-hand motifs characteristic of certain calcium binding proteins, is involved in the cation-induced activation of the enzyme (Rigutto et al., 2009). The region containing the last six transmembrane segments and the intracellular C-terminal region is homologous to these regions in the other five NADPH oxidases. In particular, this region contains the binding sites of two heme groups, one for FAD and the other for NADPH, and is the catalytic region of DUOX. Between residues 40 and 540, the ectodomain displays a low sequence similarity with both eosinophil peroxidase and TPO. The maturation of the DUOX2 protein involves the N-glycosylation of the ectodomain in the endoplasmic reticulum and a redesigning of sugar motifs in the Golgi apparatus. Transition from the endoplasmic reticulum to the Golgi and targeting to the plasma membrane require the co-expression of the DUOXA2 protein (DUOX activator 2) (Grasberger and Refetoff, 2006). Both the stability and function of DUOXA2 is totally dependent on the oxidative folding of DUOX2 (Carre et al., 2015). A functional interaction between DUOX and TPO is essential for

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regulating the level of extracellular H2O2 (Fortunato et al., 2010; Song et al., 2010). The DUOX1 protein also requires the DUOXA1 maturation factor to be active at the plasma membrane. The DUOXA1/DUOXA2 genes are located on chromosome 15 in the intergenic region of the DUOX1/DUOX2 genes and encode two transmembrane proteins of 320 and 343 aminoacids, respectively, each having 5 transmembrane segments. Mice deficient in DUOXA maturation factors
et al., 2014; or DUOX2 activity are severely hypothyroid (Donko Grasberger et al., 2012). Moreover, DUOXA2 biallelic mutations are also present in cases of permanent congenital hypothyroidism caused by iodine organification defects (Zamproni et al., 2008). Unlike the TPO and TG genes, the expression of DUOX2 is not restricted to the thyroid gland. The gene is expressed in both respiratory and gastrointestinal tracts and in salivary glands, where it plays a role in innate immunity (De Deken et al., 2014). TSH activity, via the cAMP pathway, is required to maintain a high level of DUOX2 mRNA in pig and dog thyrocytes in primary cultures (De Deken et al., 2000; Dupuy et al., 1999), but the mechanisms involved in the control of DUOX2 expression in the human thyroid are unknown. In pig thyrocytes, cAMP also increases the amount of mature DUOX2 and NADPH oxidase activity, an effect blocked by iodide (Carvalho et al., 1996; Morand et al., 2003). This inhibitory effect of iodide on the generation of H2O2 (Cardoso et al., 2001; Corvilain et al., 1988), and therefore on TPO activity, explains how excess of iodide inhibits its own organification, i.e., the WolffChaikoff effect (Wolff and Chaikoff, 1948). Protein kinase A (PKA) and PKC, which activate DUOX1 and DUOX2, respectively, via phosphorylation, are both regulated by TSH through Gs-coupled and Gq-coupled signaling pathways (Rigutto et al., 2009). Although these pathways modulate the intrinsic activity of DUOXs, Ca2þ is the primary activator of both enzymes. A decade ago, it has been shown that mice lacking the alpha subunits of Gq or G11 in their thyroids show iodine organification defect and develop hypothyroidism. Besides, these animals do not develop goiter when submitted to a goitrogenic diet, showing that in addition to thyroid function, this signaling pathway is also required for the adaptive growth of the thyroid (Kero et al., 2007). 3. Mechanisms of hormone synthesis Once they are present together at the interface of the follicular lumen and the apical plasma membrane of the thyrocytes, the molecules involved in hormonogenesis interact to produce thyroid hormones on TG. In chronological order, H2O2 oxidizes TPO, which can then oxidize iodide ions and allow their binding to tyrosyl residues of TG. Then, the oxidation and coupling of hormonogenic iodotyrosines occurs to form the iodothyronines T4 and T3 in the TG molecule (Virion, 2001). TG also contains low amounts of 3,30 ,5’triiodothyronine (rT3), 3,3’-diiodothyronine (T2) and monoiodohystidine. It is believed that TG iodination does not occur in intracellular vesicles during exocytosis; however, calveolin-1 enriched lipid rafts seem to participate in the correct addressment of the thyroid hormone machinery to the cell-colloid interface, since in caveolin-1 knockout mice thyroglobulin iodination seems to occur mainly inside the follicular cell, although the mechanism underlying these findings remains poorly defined (Senou et al., 2009). 3.1. Oxidation of the TPO by H2O2 The redox center of TPO is composed of a heme group located in a cavity of the protein and four adjacent residues, which are conserved in all animal peroxidases: histidine 239 (distal) and arginine 396, located “above” the plan of the heme outwards of the

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protein, and histidine 494 (proximal) and asparagine 579, located below the heme (Taurog, 1999). The molecule of H2O2 entering in the heme pocket interacts with histidine 239, arginine 396 and the iron atom of the heme (Fig. 1C). Two electrons are then removed from the heme, leading to “compound I” of TPO and one molecule of water. Compound I is reduced in native TPO by two electrons removed from iodide, leading to the formation of an iodinium ion (Iþ) or hypo-iodite (IOH), a very reactive species of iodine, which binds on the tyrosyl residues of TG (Fig. 1C). Compound I can also remove an electron from a molecule of iodotyrosine to produce an iodotyrosine radical, generating compound II of TPO, with only one electron missing on the heme. The return to the native form of TPO occurs via the monovalent oxidation of a second iodotyrosine, which allows the coupling reaction of two iodotyrosines to form iodothyronines. 3.2. Iodination reaction The iodination of TG is a sequential process in which TPO and TG are involved. In the thyroid gland, TG molecules have a variety of iodination degrees resulting from the functional heterogeneity of follicles and of thyrocytes that compose the same follicle. TPO oxidizes iodide in a divalent manner to produce IOH or Iþ, as described above (Taurog, 1999). However, these reactive iodine species are not just simply released into the environment. Indeed, the TG molecules that are chemically iodinated, without the participation of TPO, do not have the same iodoamino acid composition of enzymatically iodinated TG, suggesting that TPO interacts with specific tyrosyl residues of TG to iodinate them. Enzymatic iodination occurs in a maximum of 25e30 of the 132 tyrosyl residues of TG dimers. The sequential iodination of TG tyrosyl residues is dictated by its structure, which determines their reactivity. Since TPO is present as a dimer, more than one tyrosine residue might be iodinated at a time. Either chemical or peroxidase-catalyzed iodination occurs in a tyrosyl residue that will then be coupled to another iodotyrosyl residue (the hormonogenic residues). 3.3. Coupling reaction The synthesis of T3 and T4 is achieved through the transfer of an iodophenoxyl group from a MIT or DIT residue called a “donor” onto a DIT residue called an “acceptor” (Gavaret et al., 1981). This leads to an unstable quinol ether derivative that decomposes into T3 or T4 iodothyronine residues at the acceptor site and a dehydroalanine residue at the donor site (Fig. 1D). The formation of the quinol ether derivative requires prior monovalent oxidation of iodotyrosine donor and acceptor residues. As mentioned above, TPO seems to be primarily responsible for catalyzing the oxidations of iodotyrosines. It is believed that the coupling of iodothyrosines depend on the pH, since anion formation facilitates the removal of an electron under oxidative conditions, what supports the involvement of phenoxy radicals in iodothyronine synthesis (de Vijlder and den Hartog, 1998). In vivo it is mainly T4 that is formed in thyroglobulin. The structure of TG is critical for the coupling reaction, since it allows the positioning of iodotyrosine donors and acceptors in an antiparallel position, which is critical for the formation of the quinol ether derivative (Cahnmann et al., 1977). Of the 25 to 30 tyrosine residues that can be iodinated by TPO, only five to sixteen can be coupled to form 2 to 8 molecules of T4 and T3. The key acceptor residues are located at both ends of the TG chain. Other acceptor residues have been identified: tyrosines 685 and 2554. Tyrosine 130 is the main donor residue for the formation of T4 into position 5 (Targovnik, 2013). The use of these sites varies depending on the TG iodination rate, which itself depends on other physiological

conditions, such as the availability of iodide, the concentration of H2O2 and the level of stimulation of the gland by TSH (Corvilain et al., 1994). In vivo, this ensures the optimal production of thyroid hormones, even in the cases of limited iodine supply. Under normal iodine supply conditions, the thyroglobulin contain on average 2.5 residues of T4 and 0.7 residues of T3 (Dunn and Dunn, 2001), and the iodination of non-hormonogenic tyrosyl residues allows the formation of significant intra-thyroid iodine reserves that are reusable thanks to the intervention of an iodotyrosine dehalogenase (DEHAL1), whose activity is important for thyroid function (Gnidehou et al., 2004). 3.4. Iodide recycling Before thyroid hormones are released by transport across the basolateral plasma membrane domain of thyrocytes, they must be liberated from the TG core via proteolysis. The proteolysis of TG liberates not only T4 and T3, but also iodotyrosines, MIT and DIT, which are deiodinated by iodotyrosine dehalogenase (DEHAL1) that seems to be present in the apical plasma membrane, and probably in endocytotic vesicles (Gnidehou et al., 2004). After TG proteolysis, MIT and DIT are liberated, and the deiodination of these molecules by DEHAL1 is important for providing a sustained source of intrathyroidal iodide. It is believed that the catalytic site of DEHAL might face the extracellular apical surface of thyrocytes, suggesting a rapid recycling of iodide close to the organification site (Gnidehou et al., 2004). The importance of iodide recycling by DEHAL1 was confirmed by the findings that loss-of-function homozygous mutations in the DEHAL1 gene leads to high levels of urinary MIT and DIT excretion, and patients may develop goiter in the presence of a relative iodine deficiency (Afink et al., 2008; Moreno et al., 2008). 4. Thyroid hormone release from follicles The process of thyroid hormone release starts with the proteolysis of TG that can occur outside or inside the thyrocytes. The cysteine cathepsins B, K, L and S are localized at the extracellular space of the apical plasma membrane and within the endolysosomal system of thyroid cells (Jordans et al., 2009). The endocytosis of TG from the follicular lumen occurs through macro- and micropinocytosis at the apical surface of thyrocytes. Tg molecules are most likely internalized by fluid-phase endocytosis, but also by receptor-mediated endocytosis such as megalin that might be involved in the transcytosis of poorly iodinated TG (Lisi et al., 2003). When phagolysosomes are formed, thyroglobulin proteolysis occurs at a low pH inside these subcellular structures particularly by the endopeptidases cathepsins and exopeptidases (Fig. 1) (Dunn et al., 1991a, 1991b; Friedrichs et al., 2003; Jordans et al., 2009). The proteolytic products of TG are MIT, DIT, T4, T3, other iodothyronines that can be formed to a lesser extent, and amino acids. The iodination of TG tyrosyl residues are necessary for the coupling reaction to occur since only iodinated thyronines are liberated from TG. MIT and DIT are deiodinated by DEHAL1, which seems to be present at the apical plasma membrane of thyrocytes, thus recycling the liberated iodide and amino acids, as mentioned above. In the intracellular space, T4 can be deiodinated into T3 due to the presence of thyroid 5’-iodothyronine deiodinases types 1 or 2, which are expressed in rat and human thyrocytes, respectively (Abdalla and Bianco, 2014; Gereben et al., 2001). As a result, the release of T3 depends on its content in TG molecule, but also on thyroid deiodinase activity. Iodide liberated after iodotyronines deiodination might also be recycled by the thyroid. TSH stimulates TG endocytosis and thyroid iodothyronine deiodinases activities, thus increasing thyroid hormone secretion.

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T4 and T3 are released from the thyroid cell through transporters present at the basolateral plasma membrane of thyrocytes (Fig. 1). The most important transporter known to be responsible for thyroid hormone transport is the SLC16A2 monocarboxylate transporter 8 (MCT8), which can promote both uptake and efflux of TH and is involved in the release of TH from the thyroid gland (Di
pez-Espíndola et al., Cosmo et al., 2010; Friesema et al., 2004; Lo 2014; Schwartz and Stevenson, 2007). Apart from MCT8, the rat thyroid also expresses other thyroid hormone transporters, such as LAT2 (de Souza et al., 2015). Recently, iodine overload was shown to significantly reduce thyroid MCT8 expression in rats, a mechanism underlying the acute decrease of T4 release that occurs after iodine overload (de Souza et al., 2015). 5. The angiofollicular unit The thyroid follicles are highly vascularized by a fenestrated capillary network. Each angiofollicular unit (AFU) is composed of the thyrocytes that form the outer layer of the follicle, the surrounding capillaries made of endothelial cells, and pericytes. In normal follicles, the vascular bed covers approximately 20e50% of the follicle's surface. Blood flow to the thyroid increases when iodine supply decreases or TSH levels are higher; in contrast, it falls when iodine supply increases (Craps et al., 2015, 2016; Vanderstraeten et al., 2016). It is noteworthy that there is a dynamic interaction among the epithelial cells of the follicle and the endothelial cells that compose the capillary network. This interaction depends on signaling molecules that are secreted by thyrocytes or endothelial cells, such as vascular endothelial growth factor (VEGF), TGF beta, IGF-1, nitric oxide (Colin et al., 2013). An iodine deficiency increases thyroid blood flow via TSHindependent mechanisms that occur precociously and involve both endothelial cells and pericytes, as evidenced by the increased expression of the proteoglycan neuron-glial antigen 2, a marker of pericyte activation. When the intracellular iodide content decreases in thyrocytes, hypoxia-inducible factor-1 alpha (HIF-1 alpha) and VEGF expression increases, along with increases in intracellular ROS levels. VEGF activates adjacent pericytes and endothelial cells, which leads to microvascular expansion and increased blood flow under iodine-deficient conditions (Colin et al., 2013). The regulation of thyroid AFUs described above may be involved in the maintenance of thyroid hormone biosynthesis and release even under different conditions of iodine availability. Apart from the physiological importance of AFUs, the administration of saturated iodine solutions to patients with diffuse toxic goiters scheduled to undergo thyroidectomy is important for decreasing thyroid vascularization, although the mechanisms underlying these changes in AFUs still remain poorly defined. 6. Direct thyroglobulin and thyroid hormone metabolites feedback on follicles Thyroglobulin molecules in thyroid follicular lumen show different degrees of iodination, although their participation in thyroid function regulation is not well-known. In the past decades, several studies by the group of Kohn and Suzuki have shown that follicular TG regulates the expression of both thyroid transcription factors and proteins involved in iodine availability, such as NIS and pendrin (Kohn et al., 2001; Nakazato et al., 2000; Royaux et al., 2000; Suzuki et al., 1998, 2000; Suzuki and Kohn, 2006). The TG effects might involve the apical membrane asialoglycoprotein receptor (Ulianich et al., 1999), and on the other hand, the mechanism involved in TG recognition by thyrocytes to exert its negativefeedback effects in the thyroid was recently shown to depend on

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the expression of flotillin-containing lipid rafts (Luo et al., 2016). Apart from the regulation of thyroid specific genes, thyroglobulin might also be involved in the control of thyroid cell proliferation (Noguchi et al., 2010). Regarding the thyroid hormone metabolites, the in vivo inhibition of TSH secretion by 3,5-diiodothyronine has been recently described (Padron et al., 2014), which strongly suggests that this thyroid hormone metabolite does indeed interact with thyroid hormone nuclear receptors (TR), at least the TR beta isoform. In normal rats chronically treated with pharmacological doses of 3,5diiodothyronine, a strong inhibition of T4 and T3 secretion is detected even when serum TSH is still in the normal range (Padron et al., 2014). These findings indicate a direct inhibitory action of this thyroid hormone metabolite on the thyrocyte; however, one can speculate that the content of iodide that is released after its deiodination might negatively impact on thyroid function. As previously shown, thyroid follicular cells are highly sensitive to intracellular iodide levels that are negatively correlated to TSH action, so that the higher the iodine content the lower the response to TSH (Ferreira et al., 2005). However, the dose-response inhibition of T4 secretion promoted by 3,5-diiodothyronine indicates a receptormediated action, instead of an unspecific inhibition due to increased iodide content in the thyrocyte. More recently, the 3-Iodothyronamine (3-T1AM) thyroid hormone metabolite was also shown to directly act on the thyroid cell (Schanze et al., 2016). Repeated administration of 3-T1AM to mice for seven days did not interfere with the hypothalamus-pituitarythyroid (HPT) axis, while it decreased thyroidal mRNA contents of the NIS, TG, and pendrin. Also, the authors described the uptake and metabolism of 3-T1AM by thyroid PCCL3 cells. Surely, the possible effects of thyroglobulin, thyroid hormones and their metabolites in thyrocytes are of great interest to unravel the mechanisms underlying their action on the thyrocyte and their possible pathophysiological role. Funding ~o Carlos ChaThis work was supported by grants from Fundaça gas Filho de Amparo  a Pesquisa do Estado do Rio de Janeiro (26/ 010.001.252/2015) and Conselho Nacional de Desenvolvimento
gico (472630/2012-1), and from Ligue Contre le Científico e Tecnolo Cancer. References Abdalla, S.M., Bianco, A.C., 2014. Defending plasma T3 is a biological priority. Clin. Endocrinol. (Oxf) 81 (5), 633e641. Abramowicz, M.J., Targovnik, H.M., Varela, V., Cochaux, P., Krawiec, L., Pisarev, M.A., Propato, F.V., Juvenal, G., Chester, H.A., Vassart, G., 1992. Identification of a mutation in the coding sequence of the human thyroid peroxidase gene causing congenital goiter. J. Clin. Invest. 90, 1200e1204. Afink, G., Kulik, W., Overmars, H., de Randamie, J., Veenboer, T., van Cruchten, A., Craen, M., Ris-Stalpers, C., 2008. Molecular characterization of iodotyrosine dehalogenase deficiency in patients with hypothyroidism. J. Clin. Endocrinol. Metab. 93 (12), 4894e4901. Ameziane-El-Hassani, R., Morand, S., Boucher, J.L., et al., 2005. Dual oxidase-2 has an intrinsic Ca2þ-dependent H2O2-generating activity. J. Biol. Chem. 280, 30046e30054. Ameziane-El-Hassani, R., Schlumberger, M., Dupuy, C., 2016. NADPH oxidases: new actors in thyroid cancer? Nat. Rev. Endocrinol. 12, 485e494. Andrade, B.M., Araujo, R.L., Perry, R.L., Souza, E.C., Cazarin, J.M., Carvalho, D.P., Ceddia, R.B., 2011. A novel role for AMP-kinase in the regulation of the Naþ/ Iesymporter and iodide uptake in the rat thyroid gland. Am. J. Physiol. Cell Physiol. 300 (6), C1291eC1297. Arriagada, A.A., Albornoz, E., Opazo, M.C., Becerra, A., Vidal, G., Fardella, C., Michea, L., Carrasco, N., Simon, F., Elorza, A.A., Bueno, S.M., Kalergis, A.M., Riedel, C.A., 2015. Excess iodide induces an acute inhibition of the sodium/iodide symporter in thyroid male rat cells by increasing reactive oxygen species. Endocrinology 156 (4), 1540e1551. Azouzi, N., Cailloux, J., Cazarin, J.M., Knauf, J.A., Cracchiolo, J., Al Ghuzlan, A.,
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