In one study the total intrathyroidal iodine content was increased after exposure to radiocontrast media, but intrathyroidal T4 and T3 were not affected 4. It is unclear whether the mechanism s of thyroid autoregulation are similar in humans and animals, and there is no direct evidence in humans that the intrathyroidal hormonal iodine is decreased or increased by excess iodine.
In this study, humans were exposed to large doses of iodine for relatively long period of time to observe, by direct estimation of total and hormonal intrathyroidal iodine content, whether adaptation had occurred as well as the serum concentration of TSH and thyroid hormones during and after iodine withdrawal.
Patients and Methods Thirty-three euthyroid patients with normal serum thyroid autoantibody concentration without previous treatment with T4 group A who underwent operation for a single cold nodule or hyperparathyroidism were studied. Eight patients were used as untreated controls. In the remaining patients, 80— mg of iodide Lugol solution was administered daily for 15 d.
Seven patients were operated the last d 15 day on iodine and the remaining patients were divided in groups of six or seven and underwent operation at 5, 10, and 15 d after iodine discontinuation. Iodine in urine was measured before and the day of iodide withdrawal to verify compliance. Twenty-one normal volunteers group B who did not undergo operation were also studied. Serum thyroid hormones and TSH were measured before and the last day d 15 on iodine administration, and 5, 10, 15, and 20 d after iodine withdrawal.
Thyroid tissue was pulverized in liquid nitrogen and homogenized in 2 ml of cold 4 C buffer saline pH 7. From the homogenate one aliquot containing 40 mg tissue was digested with U of pronase in buffered saline. The reaction tubes were charged with nitrogen and incubated for 16 h in a shaking water bath at 37 C 5.
The hydrolysis was followed by the T4 and T3 extraction. Total tissue iodine determination. The concentration of iodine calculated by this way represents total iodine TI. The amount of iodine in intrathyroidal T4 and T3 was considered as hormonal iodine HI.
Iodide transport is energy-dependent and requires O2. Ouabain, digitoxin, and other cardiac glycosides block transport in vitro 27; Iodide uptake by thyroid cells is dependent on membrane ATPase. The rat NIS gene gives rise to a 3kb transcript with an open reading frame of 1, nucleotides encoding a polypeptide chain of amino acids.
The mature protein is a glycoprotein with an apparent molecular mass of 85kDa 31; It has 13 membrane spanning domains, with the carboxy terminus in the cytoplasm and the amino terminus located outside the cells In the model of Levy et al.
At variance with other species, three different transcripts are generated from the porcine NIS gene by alternative splicing 38 ; the main form encodes a polypeptide of amino acids as human NIS. Functional studies clearly show that NIS is responsible for most of the events previously described for iodide concentration by the thyroid. TSH exerts its regulatory action at the level of transcription through a thyroid-specific far-upstream enhancer denominated NUE NIS Upstream Enhancer that contains binding sites for the transcription factor Pax8 and a cAMP response element-like sequence.
The decrease in thyroid iodide transport resulting from excess iodide administration escape from the Wolff-Chaikoff effect, see further is related to a decrease in NIS expression 40; Reviews focus on NIS and its functional importance 50; Several mutations in the NIS gene causing defective iodide transport have been reported in humans The most commonly found mutation corresponds to a single base alteration TP in the ninth putative transmembrane domain of NIS In hypofuctioning benign or malignant tumors, the impairment of iodide transport would result from both transcriptional and post-transcriptional alterations of NIS expression Other tissues that concentrate iodide also show NIS expression, including salivary glands 67 and mammary glands 68; Iodide supply of follicular lumen involves a two-step transport process: the active transport across the basolateral plasma membrane of thyrocytes by NIS and a passive transport across the apical plasma membrane.
The protein s insuring the second step is are not yet identified. A potential iodide transporter has been proposed: pendrin 70; Pendrin, encoded by the PDS gene 72 and composed of amino acids, is expressed in different organs including kidney, inner ear and thyroid. In the thyroid, pendrin is a kDA membrane glycoprotein 73 , selectively located at the apical plasma membrane Its activity as transporter of anions including iodide has been demonstrated in different experimental systems 71; However, the implication of pendrin in thyroid iodide transport remains uncertain for several reasons.
First, there is still no direct demonstration of a pendrin-mediated efflux of iodide from thyrocytes to the follicular lumen. Second, the genetic alterations of the PDS gene found in patients with the Pendred syndrome, which lead to a loss of the anion transport activity of pendrin and to a constant and severe hearing loss, only have a moderate impact on the thyroid functioning, generally a euthyroid goiter Third, PDS knock-out mice 79 do not show any thyroid dysfunction. In summary, contrary to NIS for which the anion selectivity 25 corresponds to what was expected, the ion selectivity of thyroid pendrin remains to be elucidated.
A, immunolocalization of the human NIS protein at the basolateral plasma membrane of thyrocytes in their typical follicle organization. B, schematic representation of the membrane topology of the NIS polypeptide chain deduced from secondary structure prediction analyses C, transport of iodide from the extracellular fluid or plasma to the thyroid follicle lumen.
The uptake of iodide at the basolateral plasma membrane of thyrocytes must be active; it operates against an electrical gradient 0 - 50 mV and a concentration gradient, [ I- ]c being higher than extracellular [ I- ]. The transport of iodide from the cytoplasm to the follicle lumen should be a passive process, the electrical and concentration gradients being favorable.
Iodide that enters the thyroid remains in the free state only briefly before it is further metabolized and bound to tyrosyl residues in Tg. A major fraction of the intrathyroidal free iodide pool comes from deiodination of MIT and DIT; this iodide is either recycled within the thyroid or leaked into the circulation. Some data suggest that iodide entering the gland by active transport segregates from that generated by deiodination of Tg within the gland 82; The proportion of an iodide load that is bound varies little, despite wide shifts in daily intake.
In contrast, NIS activity is sensitive to both iodine availability and TSH stimulation, and transport rather than intrathyroidal binding is the controlling factor in making iodide available for hormonogenesis. Ductal cells of the salivary glands express NIS In the mammary gland, NIS is processed differently after translation and subjected to regulation by lactogenic stimuli As it is absent in normal non-lactating tissue, NIS may represent a marker for breast malignancy and even a possible target for radioiodine therapy The thyroid, salivary glands, and gastric mucosa share a common embryologic derivation from the primitive alimentary tract and, in each of these tissues; iodide transport is inhibited by thiocyanate, perchlorate, and cardiac glycosides.
TSH stimulates transport only in the thyroid. An active transport for iodide in the gastric mucosa has an obvious value because it provides iodine to the circulation for use in the thyroid. Active concentration by the breast helps transfer iodide to milk. Iodide concentration by the choroid plexus and salivary glands does not have any obvious physiologic benefit, but needs to be remembered for possible insights into pathways as yet undiscovered.
Iodine, particularly in the form of I2, may enter additional metabolic pathways outside the thyroid. Rats administered I2 orally showed much less circulating free iodide and much more iodine bound to proteins and lipids than did animals given iodide In another comparison of I2 versus iodide, administration of iodide to iodine-deficient rats eliminated thyroid hyperplasia much more efficiently than did I2. Additionally, I2 decreased lobular hyperplasia and periductal fibrosis in the mammary glands, while iodide increased the former and had no effect on the latter Thyroperoxidase oxidizes iodide in the presence of H2O2.
In crude thyroid homogenates, enzyme activity is associated to cell membranes. It can be solubilized using detergents such as deoxycholate or digitonin.
The enzyme activity is dependent on the association with a heme, the ferriprotoporphyrin IX or a closely related porphyrin 87; Chemical removal of the prosthetic group inactivates the enzyme, and recombination with the heme protein restores activity The apoprotein from human thyroid is not always fully saturated with its prosthetic group Some congenitally goitrous children have poor peroxidase function because the apoprotein has weak binding for the heme group Antibodies directed against the thyroid "microsomal antigen," which are present in the serum of patients with autoimmune thyroid disease AITD , led to identification of TPO.
These antibodies were found to react with proteins of kDa and to immunoprecipitate thyroid peroxidase TPO , thus identifying microsomal antigen as TPO A monoclonal antibody to purified microsomal antigen or antibodies directed againt thyroperoxidase were then used to clone human TPO Different laboratories then cloned TPO from various species: pig 99 , rat , and mouse Kimura et al.
Both forms occur in normal and abnormal human thyroid tissue. The C-terminal portion of the proteins exhibits a hydrophobic segment residues , likely corresponding to a transmembrane domain; thus, TPO has a short intracellular domain and most of the polypeptide chain is extracellular Fig.
TPO1 is active, but TPO2 appears enzymatically inactive because it does not bind heme, degrades rapidly, and fails to reach the cell surface in transfected cell lines Different degradative pathways exist for the two forms Several other TPO variants resulting from exon skipping have been identified; they appear either active or inactive Pig TPO contains amino acids 99 ; mannose-rich oligosaccharide units occupy four of its five glycosylation sites The TPO gene resides on chromosome 2p13, spans over kbp, and has 17 exons Inactivating mutations in the TPO gene are responsible for a subtype of congenital hypothyroidism characterized by thyroid dyshormonogenesis due to iodide organification defect.
More than 60 annotated mutations have been reported; most of them result in total iodide organification defect with severe and permanent hypothyroidism ; TPO synthesized on polysomes is inserted in the membrane of the endoplasmic reticulum and undergoes core glycosylation. TPO is then transported to the Golgi where it is subjected to terminal glycosylation and packaged into transport vesicles along with Tg Fig. These vesicles fuse with the apical plasma membrane in a process stimulated by TSH.
TPO delivered at the apical pole of thyrocytes exposes its catalytic site with the attached heme in the thyroid follicular lumen TPO activity is restricted to the apical membrane, but most of the thyroid TPO is intracellular, being located in the perinuclear part of the endoplasmic reticulum ; Most of this intracellular protein is incompletely or improperly folded; it contains only high mannose-type carbohydrate units, while the membrane TPO has complex carbohydrate units.
Glycosylation is essential for enzymatic activity C, hypothetical reaction scheme for TPO. H2O2 is presumed to oxidize the free enzyme with a loss of two electrons leading to the formation of complex I. A large body of older work reviewed in investigated possible sources using various in vitro models It was already suggested in that H2O2 would be produced at the apical plasma membrane of the thyrocyte by an enzyme that requires calcium and NADPH originating from the stimulation of the pentose phosphate pathway H2O2 produced by this NADPH-dependent protein is the limiting step of protein iodination and therefore of thyroid hormone synthesis when iodide supply is sufficient In human thyroid, the H2O2 production and iodination process are stimulated by the calcium-phosphatidylinositol pathway The quantity of H2O2 produced is important especially in stimulated thyrocytes; it is comparable to the ROS production of activated leukocytes.
While the activated leukocyte lives a few hours, the life of an adult thyrocyte is 7 yr ; Thus thyroid cells may be exposed to high doses of H2O2 and have to adapt to it by developing highly regulated generator and efficient protective systems.
More than twenty years passed between the initial biochemical studies and the cloning of Duox as the catalytic enzymatic core of the H2O2 thyroid generating system. By two independent molecular strategies Duox enzymes were uncovered from the thyroid. Dupuy isolated p Tox which turned out to be Duox2 lacking the first residues Simultaneously, De Deken et al cloned two cDNAs encoding NADPH oxidases using the strategy based on the functional similarities between H2O2 generation in the leukocytes and the thyroid according to the hypothesis that one of the components of the thyroid system would belong to the known gp91phox gene family and display sequence similarities with gp91phox, now called NOX2.
Duox1 and Duox2 genes are co-localized on chromosome 15q Duox1 gene is more telomeric, spans 36 kb and is composed of 35 exons; two first of them are non-coding. Duox2 spans In addition to thyroid, Duox expression is reported in several tissues: Duox1 is expressed in lung epithelia, in oocytes and Duox2 in gastrointestinal mucosa and salivary glands ; Multiple functions are attributed to Duox enzymes: airway fuid acidification , mucin secretion , wound healing ; and innate hoste defense Most of the time Duox activity is associated to a peculiar peroxidase activity like in oocyte with the ovoperoxidase involved in the fertilization process or with the lactoperoxidase in lung epithelia or in the gut ;;; Beside these killing mechanisms, Duox and H2O2 are certainly also involved in the interaction between host mucosa and bacteria to maintain mucosal homeostasis e.
In the thyroid, the specificity of the thyroid hormone machinery using Duox lays on TPO. Thus colocalization of Duox and TPO and their probable association at the apex of the thyrocyte would increase the efficiency of H2O2 producer-consumer system Onset of Duox expression study in thyroid embryonic development pointed Duox as a thyroid differentiation marker.
The proteins involved in the synthesis of thyroid hormones are expressed just after the thyroid precursor cells have completed their migration from the primitive pharynx and reached their final location around the trachea ; Until , the major obstacle for molecular studies of Duox was the lack of a suitable heterologous cell system for Duox correctly expressed at the plasma membrane in its active state.
HEK cells transfected with Duox2 generate rather small quantities of superoxide anions in a calcium-depnedent manner The reconstitution of a Duox-based functional H2O2 generating system requires a maturation factor called DuoxA.
The two human DuoxA paralogs were initially identified as thyroid specific expressed genes by in silico screenings of multiple parallel signature sequencing data bases DuoxA2 ORF spans 6 exons and encodes a amino acid protein predicted to compose five transmembrane segments, a large external loop presenting N-glycosylation sites between the second and third transmembrane helices and a C-terminal cytoplamic region Fig.
Four alternatively spliced DuoxA1 variants have been identified In heterologous systems DuoxA proteins in the absence of Duox are mainly retained in the endoplasmic reticulum. When co-transfected with Duox they cotransported with Duox to the plasma membrane where they probably form complexes.
In addition it has been shown that the type of Duox-dependent ROS poduction is dictated by defined sequences in DuoxA This means that the Duox activators promote Duox maturation but also are parts of the H2O2 generating complex ; Mice deficient in DuoxA maturation factors present a maturation defect of Duox, lacking the N-glycan processing, and a loss of H2O2 production.
These mice develop severe goitrous congenital hypothyroidism with undetectable serum T4 and high serum TSH levels The reconstitution of this functional H2O2 producing system has been useful to measure and compare the intrinsic enzymatic activities of Duox1 and Duox2 in relationship with their expression at the plasma membrane under stimulation of the major signalling pathways active in the thyroid. It has been shown that the basal activity of both isoenzymes is totally depending on calcium and functional EF-hands calcium binding motifs.
However, the two oxidase enzymatic activities are differently regulated after activation of the two main signalling cascades in the thyroid.
These results suggest that both Duox proteins could be involved in thyroid hormone synthesis by feeding H2O2 to TPO to oxidize iodide and couple iodotyrosines. From in vitro and in vivo data it has been concluded that Duox-DuoxA constitutes the major if not the unique component of the hormonogenic thyroid H2O2 generating system.
The bidirectional promoter allows the coexpression of Duox and DuoxA in the same tissue but the mechanisms regulating their transcription are not well and definitely characterized ; It has been recently shown that Th2 cytokines, IL4 and IL13, up-regulate Duox2 and DuoxA2 genes in human thyrocytes through an activation of Jak-Stat pathway opening new perspectives for a better understanding of the eventual role of Duox in autoimmune diseases They were identified in the Netherlands by neonatal screening and followed up to determine the evolution of CH with the time.
One of the patients with total organification defect TIOD presented a permanent hypothyroidism and the 8 others presented a transient hypothyroidism with a partial organification defect PIOD. Of these last 8 patients 3 harboured heterozygous nonsense or frameshift mutations QX, RX, SfsX meaning that a single defective Duox2 allele can cause haploinsufficency resulting in mild transient CH.
It is noteworthy that this hypothyroid status was limited to the neonatal period, when thyroid hormone requirement is the highest, and was not detectable in adulthood since adult heterozygotes in these families presented normal TSH serum levels. No mutation was detected in Duox1. With the increasing number of reported Duox2 mutations in CH, it becomes more and more difficult to make the correlation between genotype and phenotype as initially described.
Patients with compound heterozygous missense RW and a nonsense mutation RX , leading to a presumed non functional protein showed PIOD with mild and persistent hyperthyrotropinemia. This suggests that Duox1 can compensate at least partially for the defect in Duox2 Varela et al.. The phenotype-genotype correlation suggested by the work of Moreno et al. Maruo et al.
Three of them had low free T4 at birth, mild thyroid enlargement. The thyroid hormone replacement therapy ceased to be necessary by 9yr of age A French-Canadian patient with a transient CH initially detected by neonatal screening presented a compound heterozygozity for a hemizygous missense mutation GS inherited from the father and a deletion removing the part of the gene coding for the catalytic core of Duox2 inherited from the mother.
In vitro test proved that the missense mutant protein was totally inactive This case and others reported later provide further evidence that permanent or transient nature of CH is not directly related to the number of inactivated Duox2 alleles The first homozygous nonsense mutation in DuoxA2 YX that resulted in a non-functional protein tested in vitro has been found to be responsible of a permanent mild CH in a Chinese patient with a dyshormonogenic goiter ; The variety of observerd phenotypes associated with Duox2 and now DuoxA2 mutations suggest that the manifestation of Duox2 defects could likely be influenced by the environmental factors like iodine intake or by the activation of Duox1 or DuoxA1 in peculiar circumstances.
B, Schematic representation of the predicted structure of DuoxA from Its main function is to provide the polypeptide backbone for synthesis and storage of thyroid hormones It also offers a convenient depot for iodine storage and retrieval when external iodine availability is scarce or uneven.
Neosynthesised Tg polypeptide chains entering the lumen of the rough endoplasmic reticulum RER are subjected to core glycosylation, dimerise and are transferred to the Golgi where they undergo terminal glycosylation Fig. Iodination and hormone formation of Tg occur at the apical plasma membrane-lumen boundary and the mature hormone-containing molecules are stored in the follicular lumen, where they make up the bulk of the thyroid follicle colloid content.
Inside the lumen of RER, newly-synthesized proteins undergo core glycosylation and by interacting with chaperones acquire their conformation. Proteins are then transported to the Golgi apparatus G , where terminal glycosylation and other post-translational reactions take place. In the Trans-Golgi network TGN , mature proteins undergo sorting processes and are packed into transport vesicles. The vesicles carrying soluble proteins inside the vesicle and membrane proteins as integral vesicle membrane protein deliver them at the appropriate plasma membrane domain: the apical domain 1 and 2 or the basolateral domain 4.
Vesicles carrying lysosomal enzymes 3 conveyed their content to prelysosomes or late endosomes LE and lysosomes L. The Tg peptide chain derives from a gene of more than kbp located on chromosome 8 in humans. The human Tg gene consisting of 48 exons gives rise to a 8. The primary structure deduced from cDNA is also known for bovine, rat, and mouse The biochemical traits of human Tg have been reviewed in Such domains have been found to be potent inhibitors of cysteine proteases This finding might be of importance, because these proteases are active in Tg proteolysis see below.
It has been suggested that this region of the Tg molecule may modulate its own degradation and hormone release In the Tg-type 1 repeats, cysteine and proline residues are found in constant position; they may have an important role in the tridimensional structure of the protein. The proximal region of the C-terminal half portion of Tg contains five repeats of another type of cysteine-rich motives. The presence of a high number of cysteine residues in Tg, involved for most of them in disulfide bonds, probably gives rise to peculiar structural constraints.
The C-terminal portion of Tg is homologous with acetylcholinesterases Because binding to cell membranes is one feature of acetylcholinesterases, perhaps Tg C-terminus has a similar role.
It was reported that the acetylcholinesterase-homology region of Tg could function as a dimerization domain ; Furthermore, three highly conserved thioredoxin boxes have been identified in mammalian Tg between residues 1, and 1,; these boxes might be involved in disulfide bond formation leading to intermolecular cross-linking of Tg molecules inside the follicle lumen Hydrogen peroxide might be a regulatory factor of Tg expression, based on experimental work showing increased Tg promoter activity with reduced Pax-8 and TTF-1 Maturation of the Tg polypeptide chain begins while still on the RER.
It undergoes core glycosylation and then monomers fold into stable dimers. Arvan and co-workers have mapped this process and emphasize the role of molecular chaperones. The latter are essential for folding the new Tg molecules, and those that are folded improperly are not allowed to proceed further. Only Tg molecules that pass this quality control system unscathed can proceed towards the secretory pathway. Glycosylation is a key event in Tg maturation.
Human Tg may contain four different types of carbohydrate units. The "polymannose" units consist only of mannose and N-acetylglucosamine. The "complex unit" has a core of three mannose residues with several chains of N-acetylglucosamine, galactose, and fucose or sialic acid extending from them. Both these types of unit are common in glycoproteins and are linked to peptide through an asparagine-N-acetylglucosamine bond. About three quarters of the potential N-glycosylation sites in human Tg are occupied, mostly with the complex unit Two additional units have been found in human Tg; one contains galactosamine and is linked to the hydroxyl group of serine, the other is a chondroitin sulfate unit containing galactosamine and glucuronic acid Their Tg shows abnormal folding and decreased export from the ER in association with increased levels of several molecular chaperones.
Correction of this defect by site-directed mutagenesis returned Tg export to normal in transfected cells. Other examples are cystic fibrosis, osteogenesis imperfecta, familial neurohypophyseal diabetes insipidus, insulin receptor defect, growth hormone receptor defect, and a variety of lipid disordersKimura et al. Four alternatively spliced DuoxA1 variants have been identified Beside these killing mechanisms, Duox and H2O2 are certainly also involved in the interaction between host mucosa and bacteria to maintain mucosal homeostasis e. At secretion with other development, three different transcripts are generated from the very NIS gene by alternative splicing 38 ; the main form encodes a polypeptide of similar acids as human NIS. Cowboy: In 33 euthyroid patients with robust Regeneration pat barker essays nodule or hyperparathyroidism, Lugol acronym 80 mg iodine was administered for 15 d before delivery. In the Tg-type 1 repeats, cysteine and solid residues are found in particular position; they may have an important goal in the tridimensional structure of the food. C and D, alabama images of the top C and the bottom D of the mystery after 2hr of incubation. In defamation to synthesis, Duox expression is thyroid in several hours: Duox1 is expressed in lung ailments, in oocytes and Duox2 in gastrointestinal mucosa and experienced hormones ; The readers involved in the processing of thyroid hormones are expressed just after the developmental precursor cells pdf told their migration from the primitive informality and reached their assignment location around the trachea ; Reviews focus on NIS and its validity importance 50.
Rats administered I2 orally showed much less circulating free iodide and much more iodine bound to proteins and lipids than did animals given iodide Design: In 33 euthyroid patients with single thyroid nodule or hyperparathyroidism, Lugol solution 80 mg iodine was administered for 15 d before operation.
The radioautographs indicated the presence of iodide primarily over the cells at these early time intervals. Next, the two reactants form compound II, which is necessary for the coupling reaction to make thyroid hormones. From Pitt-Rivers, R. The exopeptidase lysosomal dipeptidase II may also be involved in release of free T4, but from a site in Tg other than residue 5 B, schematic representation of the membrane topology of the NIS polypeptide chain deduced from secondary structure prediction analyses
Its activity as transporter of anions including iodide has been demonstrated in different experimental systems 71; Other examples are cystic fibrosis, osteogenesis imperfecta, familial neurohypophyseal diabetes insipidus, insulin receptor defect, growth hormone receptor defect, and a variety of lipid disorders Finally, free thyroid hormones apparently diffuse out of lysosomes, through the basal plasma membrane of the cell, and into blood where they quickly bind to carrier proteins for transport to target cells.
Stored Tg molecules undergo iodination and hormone formation reactions at the apical plasma membrane-lumen boundary , where TPO and H2O2 generating system reside. Arvan and co-workers have mapped this process and emphasize the role of molecular chaperones. It has been shown that the basal activity of both isoenzymes is totally depending on calcium and functional EF-hands calcium binding motifs. This is an iodinated albumin, shown to be serum albumin that is iodinated in the thyroid
Small increments of iodine go first to tyrosyl residues , , , , , and 5, in that order. Until , the major obstacle for molecular studies of Duox was the lack of a suitable heterologous cell system for Duox correctly expressed at the plasma membrane in its active state.
The other Tg molecules with no or a low hormone content, present in early apical endosomes, enter either of the two following routes; they are recycled back into the follicle lumen through a direct vesicular transport towards the apical plasma membrane or via a two-step vesicular transport to the Golgi apparatus and then to the apical plasma membrane Thyroperoxidase oxidizes iodide in the presence of H2O2. Starting from highly purified preparations of thyroid lysosomes, Rousset et al.