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Institut für Experimentelle Endokrinologie (J.K.), Charité Universitätsmedizin Berlin, Humboldt Universität, D-10098 Berlin, Germany; Experimentelle und Klinische Osteologie (F.J.), Orthopädische Universitätsklinik, D-97074 Würzburg, Germany; and Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (B.C., J.E.D.), Université Libre de Bruxelles, Campus Hopital Erasme, B-1070 Bruxelles, Belgium
Correspondence: Address all correspondence and requests for reprints to: Prof. Dr. Josef Köhrle, Institut für Experimentelle Endokrinologie, Charité, Humboldt Universität zu Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany. E-mail: josef.koehrle{at}charite.de
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Abstract |
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 Top Abstract I. Historical AspectsII. Biosynthesis and Degradation...III. Recently Discovered...IV. Hormonal Regulation of...V. Selenium, Cell Defense,...VI. Selenoproteins and the...VII. Selenium and the...I. Selenium, the hormonal...J. Selenoproteins and the...References |
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I. Historical Aspects |
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TopAbstractI. Historical AspectsII. Biosynthesis and Degradation...III. Recently Discovered...IV. Hormonal Regulation of...V. Selenium, Cell Defense,...VI. Selenoproteins and the...VII. Selenium and the...I. Selenium, the hormonal...J. Selenoproteins and the...References |
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). The essential role of selenoproteins in the endocrine network besides the thyroid axis is becoming evident: they are involved in peroxide degradation, cellular redox and transcription regulation, thyroid hormone deiodination, spermatogenesis, and several additional, still unknown biochemical pathways. Recently, the first mutations in selenoproteins [SECIS binding protein (SBP) 2 and SEPN1] have been linked to human diseases, i.e., disturbances of thyroid hormone metabolism (22) and a rare form of congenital muscle dystrophy (23, 24).
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II. Biosynthesis and Degradation of Eukaryotic Selenoproteins |
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TopAbstractI. Historical AspectsII. Biosynthesis and Degradation...III. Recently Discovered...IV. Hormonal Regulation of...V. Selenium, Cell Defense,...VI. Selenoproteins and the...VII. Selenium and the...I. Selenium, the hormonal...J. Selenoproteins and the...References |
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In contrast, the biosynthesis of the 21st amino acid, Sec, and its cotranslational incorporation into specific proteins are highly regulated (25). The codon UGA not only acts as an opal stop codon during translation, but also encodes the translational incorporation of Sec into proteins when the mRNA contains a distinct hairpin mRNA sequence downstream of the UGA codon in its 3'-untranslated region (3'-utr) (Fig. 1). This Sec insertion sequence (SECIS), or Sec translation element, prevents termination of the translation by competing for release factors that would otherwise lead to disassembly of the mRNA-ribosomal complex (7, 26). In eukaryotes, the SECIS structure recruits the SBP2 (27) and binds the Sec-specific elongation factor (EFSec) loaded with its tRNASec. In prokaryotes, but not archeae, SelB exerts the function of these two proteins (28, 29, 30, 31). Several other candidate proteins binding to SECIS elements are currently being investigated (32, 33). The SBP2 specifically binds selenoprotein mRNAs, with no known preferences for individual SECIS structures. SBP2 probably prevents termination of protein translation at the UGA codon (34) but does not protect -tRNA (35). Mutations in SBP2 lead to impaired Se status and reduced expression of several selenoproteins, including plasma GPx (pGPx), selenoprotein P (SePP), and type II 5'-deiodinase (D2), resulting in abnormal thyroid hormone metabolism (22).
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The synthesis of Sec occurs in a complex reaction by pyridoxal phosphate cofactor-dependent selenophosphate incorporation into the serine of the serine-loaded tRNASer(Sec) via the enzyme Sec-tRNA synthase. The biosynthesis of selenophosphate is catalyzed by SelD. One form of this enzyme, encoded by the SelD2 or sps2 gene, is by itself a Sec-containing protein (41), although the role of the non-Sec form SelD1 is still controversial (42, 43). Se supply controls the first step in the biosynthesis of Sec-containing proteins. The components required for cotranslational Sec incorporation into proteins are homologous to the systems so far defined in prokarya, archeae, and Drosophila (37, 44, 45, 46, 47, 48). Disruption of selenoprotein biosynthesis in Drosophila by inactivation of SelD affects cell proliferation and development (48). Mutants lacking the translation elongation factor SelB/EFSec are viable and fertile, even in the complete absence of selenoprotein biosynthesis (49). In contrast to the prokaryotic selenoprotein mRNA, in which the SECIS element lies immediately downstream of the UGA codon, in eukaryotes the SECIS element is located up to 6 kB downstream of the UGA codon in the 3'-utr. Translation of eukaryotic Sec-containing proteins, albeit at low efficiency, can be achieved in cell culture systems from cotransfected expression plasmids (50, 51). It is improved with tRNA(Ser)Sec and SelD2. In proteins, Sec exerts its prominent and specific functions due to its high redox potential and the low pKa value (5.7) of its selenol (-SeH) group compared with that of most of the sulfhydryl (-SH) groups of cysteine residues (pKa 
8.5). The -SeH group of Sec proteins is readily oxidized by H2O2 similar to some few acidic cysteine residues in selected proteins (52).
Sec degradation is catalyzed by pyridoxal-5'-phosphate-dependent Sec lyase, which is highly specific for Sec and does not metabolize cysteine (53). It forms alanine from Sec and recycles Se probably as elemental Se that may then be cotranslationally incorporated into tRNASer(Sec) by SelD2. Sec lyase is distantly related to the Escherichia coli enzyme NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters (42, 53). In contrast, selenomethionine is metabolized by the same enzymes handling methionine.
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III. Recently Discovered Eukaryotic Selenoproteins |
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TopAbstractI. Historical AspectsII. Biosynthesis and Degradation...III. Recently Discovered...IV. Hormonal Regulation of...V. Selenium, Cell Defense,...VI. Selenoproteins and the...VII. Selenium and the...I. Selenium, the hormonal...J. Selenoproteins and the...References |
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and Fig. 2). A fifth, highly homologous non-seleno-GPx (GPx-5), which does not contain Sec and is controlled by androgens, has been described in epididymis and testes of rodents and monkeys (54, 55, 56), in bovine keratinocytes, and in eyes and human skin (57, 58). Apparently, the mRNA of this GPx-5 is not translated into a functional protein in human epididymis (59). A sixth GPx form, highly abundant in the testes, which has no homolog in the mouse, has recently been identified in the systematic in silico screen for selenoproteins in the human genome (20).
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). The cloning of the D1 gene revealed the structural elements required for translation and identification of the SECIS structure (13, 60). Recently, another Se-containing enzyme family of three members, the mammalian thioredoxin (Trx) reductases (TrxRs) (Fig. 4), was identified. Their prokaryotic orthologs do not contain Se (61).
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) whose biological functions are unknown (PES, p15, SelH, SelI, SelK, SelM, SelN, SelO, SelR, SelS, SelT, SelV, SelX, and SelZ) or not yet fully established (SelP, SelW) have been characterized or cloned during the last 5 yr (20, 62, 63, 64, 65). Most of these proteins appear to be involved in redox reactions, such as the methionine sulfoxide reductase B (SelR) (66). They metabolize unusual substrates or contribute to the reduction of reactive oxygen species (ROS) such as peroxides or peroxinitrite. Sep15, highly expressed in human thyroid, prostate, and testes, seems to be closely associated with the endoplasmic reticulum resident enzyme UDP-glucose-glycoprotein-glucosyltransferase. It may participate in quality control of misfolded, newly synthetized proteins (67). In addition, Sep15 is involved in growth inhibition and apoptosis (68). The SEPN1 gene has been discovered by in silico cloning based on the SECIS motive (63). It is the first link of a selenoprotein to a rare human congenital disease, the rigid spine syndrome, a form of muscular dystrophy (23, 69). SePP, a glycosylated human plasma protein containing up to 70% of plasma Se, is an unusual selenoprotein that contains up to 10 Sec residues per molecule in most mammals, 12 in bovine species, and up to 17 in zebrafish. Recent research implies both a low efficiency peroxidase function and the binding of heavy metals such as mercury or cadmium (70, 71). SePP, which has strong affinity for heparin, avidly binds to the endothelial surface and might protect the endothelium from oxidative damage (18, 72, 73). Secreted SePP mainly is of hepatic origin, but several tissues express its mRNA. If translated at adequate Se supply, SePP might function in extracellular compartments or at cellular surfaces as a component of cellular antioxidative defense systems and actively scavenge peroxinitrite (18, 74, 75, 76, 77). The successful generation of two viable mouse knockout models for SePP (78, 79) provides strong evidence for the initial hypothesis that SePP serves as a Se transport and delivery protein for other tissues. In these models nutritive Se accumulates in the liver, the main site of SePP synthesis, whereas other tissues including brain show markedly lower Se content and activities of selenoproteins. This may induce ataxia and impaired growth (78) due to disturbance of the GH axis (see Section III.B). Increased selenite in drinking water can rescue the mouse phenotype (33, 80). Other cytoplasmic and plasma Se-binding proteins are known (e.g., SP56) (81).
According to metabolic labeling experiments with 75-selenite in severely Se-depleted rats, 2-D gel electrophoretic autoradiographic patterns reveal more than 25 individual selenoproteins (82). These might represent transcripts with different start sites and promoters and translation products of alternative splice forms of the 25 human or 24 mouse selenoprotein-encoding genes. Several new genes encoding putative selenoproteins are currently being characterized (Table 1) (6, 20, 62, 63, 83, 84). The proteins were identified by the in silico approach based on comparative genomics and characteristic sequence and structure motifs of selenoprotein-encoding genes.
B. Preferential selenium supply of the vital endocrine organs during deficiency and repletion
A general observation during Se depletion was the retention or redistribution of Se to the brain, the endocrine organs, and the reproductive organs, whereas liver, muscle, skin, and other large tissues rapidly lose their Se (85). In these tissues, Se is rapidly mobilized from cellular cGPx stores, whereas expression of other selenoproteins such as phospholipid hydroperoxide GPx (PHGPx) and GI-GPx, the deiodinases (especially type II and type III), and TrxRs is hardly affected or may even be increased (type I 5'D). Uptake of Se compounds into cells is assumed to occur via anion transporters (86, 87, 88, 89, 90, 91). Selenite is assumed to be transported by the sulfate transporter (92, 93). In the hierarchy of biosynthesis of selenoproteins during Se repletion, some mRNAs are preferentially translated into selenoproteins. This preference may be directed by the two forms of SECIS elements (29, 94, 95). Full expression of SePP requires a greater Se intake than does full expression of pGPx. This suggests that SePP is a better indicator of Se nutritional status than is GPx (96). In general, those proteins residing high in the hierarchy of Se retention during Se depletion also appear to lead in the priority for repletion (97, 98, 99, 100, 101, 102).
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IV. Hormonal Regulation of the Thioredoxin/Thioredoxin Reductase System |
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TopAbstractI. Historical AspectsII. Biosynthesis and Degradation...III. Recently Discovered...IV. Hormonal Regulation of...V. Selenium, Cell Defense,...VI. Selenoproteins and the...VII. Selenium and the...I. Selenium, the hormonal...J. Selenoproteins and the...References |
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B. Biochemistry and structure of thioredoxin reductase
Mammalian TrxRs are flavin adenine dinucleotide-containing flavoproteins using nicotinamide adenine dinucleotide phosphate (NADPH) + H+ as their cofactor system, and therefore the pentose phosphates cycle as reducing pathway. Their active site contains a reduced pair of cysteine residues in the N-terminal region. They differ from glutathione reductases by a conserved C-terminal GCUG sequence (U stands for Sec) that is essential for enzyme activity. Lack of Sec incorporation and premature termination of the polypeptide chain at the C residue in the absence of adequate Se supply produces an inactive protein (110, 111). A similar C-terminal structure has also been identified in one of the TrxR proteins of Caenorhabditis elegans, but not in a second TrxR enzyme more similar to the prokaryotic TrxR without the Sec residue (127). This essential penultimate Sec residue in mammalian TrxR may act as a cellular redox sensor for regulation of gene expression (128) or in apoptosis (129). TrxRs are members of the pyridine nucleotide-disulfide oxidoreductase family, which includes glutathione reductase, lipoamide dehydrogenase, and mercuric ion reductase.
The discovery of several TrxR genes and their splice variants (at least three isoenzymes: TrxR1, -2, and -3) (61, 117) suggests a specific compartmentalized and fine-tuned regulation of redox-sensitive proteins and signaling cascades (130).
C. Thioredoxin reductase and thioredoxin are involved in signal transduction and regulation of gene expression
1. Redox-regulated transcription factors.
Several of the redox reactions modulated via the Se Trx/TrxR system are mediated through the cellular redox/DNA repair protein redox factor 1 (Ref-1). This stimulates DNA-binding activity of several classes of redox-regulated transcription factors, such as activator protein 1 (AP-1), nuclear factor 
B (NFB), Myb, Ets, and the redox-sensitive nuclear receptor family (131, 132, 133, 134, 135). Several signals have been found to translocate TrxR into the nuclear compartment where preformed Ref-1 and TrxR1 exist. Interaction with transcription factors AP-1 and p53 may result. The signals include: activation of protein kinase C (PKC), stimulation by cis-diaminedichloroplatinum II, oxidative stress, cytokines, lipopolysaccharide, or UV irradiation (133, 136, 137, 138, 139) (Fig. 5).
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B (140), AP-1 (jun and fos) (141), Sp1 (142), glucocorticoids (143), estradiol (134, 135, 144), retinoids (145), and other nuclear receptor systems, Janus-activated kinases, MAPK, protein-tyrosine phosphatases (128), thyroid transcription factor-1 (TTF-1), and p53 (136, 143). The Sec residue of TrxR and other selenoproteins could act as a sensor for cellular ROS (128), which themselves regulate the phosphorylation cascades.
2. Modulation of intracellular signaling cascades by TrxR/Trx.
Involvement of the TrxR/Trx system in transcription regulation and proliferation has been demonstrated for several cell types. In A431 cells, epidermal growth factor (EGF) leads to H2O2 and ROS production, similar to direct H2O2 stimulation, with oxidation of the Sec residue of TrxR and oxidative inhibition of phosphotyrosine phosphatase 1B and tyrosine phosphorylation of proteins. Trx reduces phosphotyrosine phosphatase 1B and regenerates the system. Prolonged incubation with EGF or H2O2 induces neosynthesis of TrxR with its regulatory consequences (128). Trx1 is also induced by many variants of "stress," such as UV radiation, x-rays, viral infection, oxidative stress, and several cytostatic (cis-platinum II) compounds or redox-active agents (136). Selenite inhibits UVB-induced cell death (146) and cell death enzymes, and these effects are reversed by dithiothreitol (DTT) or ß-mercaptoethanol compounds.
3. TrxR/Trx-modulated effects on proliferation and tissue specific gene expression.
Trx enhances, whereas oxidants inhibit, the effect of various transcription factors and nuclear receptors: the estrogen receptor 
and glucocorticoid receptor (135, 147). Dominant negative Trx mutants or antisense Trx plasmids inhibit breast tumor cell growth and revert the transformed phenotype (148, 149). Trx also augments redox-sensitive DNA binding activity of the tumor suppressor protein p53, (also activated by Ref-1), and thus stimulates p21 production. A transdominant inhibitory mutant of Trx suppressed the effects of Trx on Ref-1, p53, and p21 activation (136).
Alterations of intracellular glutathione levels have been shown to differentially affect gene expression in the differentiated thyroid cell line FRTL-5 (150). Depletion of intracellular glutathione by treatment of cells with the inhibitor of 
-glutamylcysteine-synthetase butylsulfoxime specifically impairs the transactivation potencies of the thyroid-enriched transcription factors Pax-8 and more so of TTF-1 on the promoters of thyroglobulin (Tg) and to a lesser extent thyroperoxidase (TPO) genes. Se may influence thyroid gene expression directly via selenoprotein or indirectly through modulation of the cellular redox status.
4. Additional substrates of the TrxR/Trx system.
Apart from its action on cellular redox components as an antioxidative system, TrxR appears to be involved in reduction of Trx peroxidase and peroxiredoxins, enzymes that degrade H2O2 to water (151, 152, 153). Furthermore, TrxR and Trx supply reducing equivalents for cellular redox-regulated enzymes such as ribonucleotide reductase, a factor in DNA biosynthesis. Other TrxR substrates include several drugs, dehydroascorbic acid and ascorbyl free radical, vitamin K3, lipoic acid and lipid hydroperoxides, and NK-lysine, a cytotoxic peptide produced by natural killer cells (154, 155). This broad specificity is unusual but might be due to the C-terminal penultimate exposed Sec residue of TrxR (156, 157).
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V. Selenium, Cell Defense, and Thyroid Pathology |
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TopAbstractI. Historical AspectsII. Biosynthesis and Degradation...III. Recently Discovered...IV. Hormonal Regulation of...V. Selenium, Cell Defense,...VI. Selenoproteins and the...VII. Selenium and the...I. Selenium, the hormonal...J. Selenoproteins and the...References |
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). The former show, aside from their mental retardation, signs of severe hypothyroidism, developmental retardation (i.e., dwarfism), myxedema, and—unlike the normal population of the area—they present no goiter. Neurological cretins are almost normally developed, do not exhibit signs of hypothyroidism, have goiters as the rest of the population, but have various neurological deficits. These sometimes include deaf-mutism. Pure forms of myxedematous cretinism predominate in Central Africa, but there are neurological cretins and myxedematous cretins with neurological defects. In other endemic regions like New Guinea or in South America, only neurological cretinism is detected. Both forms, along with intermediates, coexist in India (159, 160). The concept that the two syndromes are linked to a common cause, i.e., iodine deficiency, is now well accepted (161, 162, 163, 164). Neurological cretinism stems from deficient thyroid hormone in early fetal development (165, 166, 167). Myxedematous cretinism is associated with thyroid insufficiency during late pregnancy and early infancy (159, 168, 169). The distinct geographical distribution of the two forms of cretinism, as well as their different phenotypes, suggests that other factors are involved. Among these are: 1) autoimmune disorders and TSH inhibitory antibodies (170, 171); 2) nutritional habits like cassava consumption and the thiocyanate overload that ensues, impeding iodide trapping (172); 3) trace element deficiencies like zinc, copper, manganese, iron, and Se (173, 174, 175) through their involvement in enzymes implicated in cell defenses; 4) vitamin A and E deficiencies also involved in cell defenses against free radical attacks (176); and 5) enzyme deficiencies like superoxide dismutase deficiency or glucose-6-phosphate-dehydrogenase, possibly leading to decreased efficacy in glutathione reduction (176).
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2. Myxedematous cretinism resulting from thyroid destruction in early life.
Myxedematous cretins are hypothyroid as shown by their clinical (skin texture, sensitivity to the cold, slowness, slow reflexes), biological (low thyroid hormone levels), and radiological characteristics (bone development retardation) (178). Signs of developmental (height) and mental retardation are proportional to the degree of hypothyroidism, which suggests a causal relationship (162, 163, 164). All these characteristics are similar to those of sporadic congenital hypothyroidism.
Primary thyroid insufficiency causes hypothyroidism as shown by the high serum TSH levels and the absence of thyroid response to additional TSH administration. The insufficiency itself results from thyroid atrophy, presumably from thyroid damage, and as demonstrated by the absence of goiter, a low radioiodine uptake, a reduced thyroid activity under radioiodide scanning, and a rapid radioiodine turnover (161, 162, 164, 179). A unique autopsied thyroid of a Congolese cretin showed severe fibrosis with a few overactive follicles constrained in a fibrotic network (Fig. 6). Thyroid destruction is a slow process (169, 180). It affects the population well beyond the pathology of myxedematous cretinism (181).
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3. Thyroid fibrosis as a common feature of endemic cretinism and goiter.
The description of a thyroid destruction process in an area of endemic goiter, i.e., thyroid hyperplasia, may appear paradoxical. However, the same pathological process can be proposed to explain the coexistence of goitrous subjects with myxedematous subjects having a destroyed thyroid. Iodine deficiency leads to high TSH, thyroid proliferation, and goiter formation to such an extent that goiter by itself impairs efficient use of iodine and thyroid hormone synthesis and thus becomes a maladaptation to iodine deficiency (182). In the peculiar condition of Central Africa (Se deficiency, thiocyanate exposure) pronounced TSH stimulation leads to significant thyroid necrosis, which further increases thyroid proliferation. Thyroid necrosis promotes fibrosis within the wounded thyroid, which may impede proliferation and tissue repair (183). As a result, the evolution of hypothyroid subjects to develop a big goiter or to experience gland destruction depends on which of the two processes, proliferation or fibrosis, wins. The destruction process affects the population on a large scale. In severe cases people become deeply hypothyroid and develop myxedema. Thyroid damage within the rest of the population decreases the efficacy of iodine supplementation programs (181) by decreasing iodide trapping and impairing the adaptive mechanisms (162). Although some myxedematous cretins may improve their thyroid status and even resume a euthyroid status under high iodine supplementation, others may not (168, 169, 180, 184). The fibrotic process may be important for the irreversibility of thyroid destruction by impeding repair through the cell proliferation that follows necrosis in a process akin to liver cirrhosis (179).
4. Biochemical relation of Se deficiency to thyroid destruction.
Other trace element deficiencies could act together with iodine deficiency in inducing thyroid destruction (173). Trace elements involved in GPx and superoxide dismutases enzymes activities—i.e., Se, magnesium, copper, and zinc—were lacking in Idjw Island (Central Africa) in two comparably, iodine-deficient areas, one with prevalent myxedematous cretinism, the other without. Only Se deficiency correlated both with the geology and with the distribution of myxedematous cretinism.
The underlying hypothesis was that the thyroid gland, which produces H2O2 for thyroid hormone synthesis, is exposed to free radical damage if H2O2 is not properly reduced to H2O by intracellular defense mechanisms or during the hormone synthesis process (185). H2O2 is essential for the TPO enzyme in the process of iodide oxidation. In the human thyroid gland, the H2O2 generating system is under the control of TSH through the stimulation of the phospholipase PIP2-IP3-Ca2+cascade (186, 187). When iodine supply is sufficient, this H2O2 generation is thought to be the limiting step for thyroid hormone synthesis; H2O2 is reduced to H2O during the process of synthesis. However, the KM of TPO for H2O2 is high, and much higher amounts of H2O2 are produced than consumed by the iodination process (188, 189), potentially exposing the thyroid gland to free radical damage (185). The H2O2 exposure is greatest with maximal TSH stimulation. In human thyroid slices, high levels of TSH increase the generation of H2O2 up to 13 times the level produced by activated leukocytes (188, 189). TSH secretion is acutely stimulated at birth with the postnatal TSH rise and chronically at all times under iodine deficiency conditions.
Protection against H2O2 and resulting free radicals entails vitamins C and E and enzymes such as catalase, superoxide dismutase, and Se-containing enzymes. Originally, GPx was the only identified selenoenzyme (4, 5, 173, 190). However, other Se-dependent enzymes are present in the thyroid and involved in antioxidant defenses (4, 74, 107, 191, 192, 193). PHGPx is an example (194, 195). Thus, iodine deficiency increases H2O2 generation, whereas Se deficiency decreases H2O2 disposal.
5. Epidemiological studies.
Epidemiological surveys suggested concomitant Se and iodine deficiencies where myxedematous cretinism is highly prevalent in Central Africa, i.e., in the goiter belt crossing the Congo/Zaire (173, 176, 196). However, a similar association of iodine and Se deficiency in Tibet and in China does not lead to myxedematous endemic cretinism. Thus, iodine and Se deficiencies do not appear sufficient for thyroid destruction. Another important factor in the pathogenesis of endemic goiter in Africa had already been well identified and documented: thiocyanate. Thiocyanate overload results from cassava consumption, a staple in Central Africa, but not Tibet and China. Cassava roots contain the cyanogenic glucoside linamarin (197, 198). Linamarin metabolism releases cyanide, which is detoxified to thiocyanate, a known goitrogen (198). It competes with iodide for trapping by the sodium iodide symporter and for oxidation by the TPO (199). Thiocyanate induces both a release of iodide from the thyroid cell and a decrease of thyroid hormone synthesis. Experimental and epidemiological studies have shown that thiocyanate overload aggravates the severity of iodine deficiency and worsens its outcome (177, 198, 200). However, the common association of these two factors in Central Africa is not sufficient to explain the more restricted prevalence of myxedematous cretinism (196).
6. Se deficiency increases the sensitivity to necrosis in various models.
Neither in the thyroid nor in other tissues have experiments shown a deficiency restricted to Se (177, 201, 202). Obvious necroses have only been documented when Se deficiency combines with vitamin E deficiency or additional stressors that lead to an additional decrease in cell defense (202, 203, 204, 205, 206, 207). Under these conditions, agents such as paraquat, diquat, or carbon tetrachloride induce necrosis in the liver (202, 203, 204, 205, 206, 207).
Myopathy has also been reported in Se-deficient calves that have exercised to excess (208, 209). In the cardiomyopathy of Keshan disease described in China in association with Se deficiency, the proposed additional stress is more complex. Se deficiency would first facilitate somatic viral mutations in the coxsackie B3 virus, which in turn would become more aggressive for the Se-deficient heart and induce necrosis (202, 210). Water pollutants, i.e., fulvic acid, leading to superoxide production, can induce joint damage in mice (202, 211, 212, 213). In this disease, aflatoxins may also play a role. Moreover, a statistical relation between iodine deficiency in association with Se deficiency has been recently shown in the Kashin-Beck disease, suggesting that iodine deficiency plays a role in the etiology (214). Thus, Se deficiency per se, in the thyroid as in other tissues, is not sufficient for, but facilitates tissue destruction.
B. Experimental thyroid model
Experiments in rats failed to reproduce major thyroid damage from the single association of Se and iodine deficiencies (15). However, Se deficiency increases the sensitivity of the thyroid gland to necrosis caused by iodide overload in iodine-deficient thyroid glands (215, 216, 217, 218, 219, 220). Another group failed to repeat this finding (221). Se deficiency increases the inflammatory reaction initiated by iodide overload that then evolves to fibrosis, whereas the non-Se-deficient thyroid exhibits no fibrosis (222). Fibrosis was associated with increased fibroblast proliferation and decreased thyroid follicular cell proliferation (222). TGFß was prominent in thyroid macrophages in Se deficiency and was proposed to be responsible for both effects (177, 183). Indeed, TGFß stimulates the proliferation of fibroblasts and promotes fibrosis, and on the other hand it impairs TSH-induced proliferation (223). TGFß-blocking antibodies do the reverse, blocking the evolution of the thyroid to fibrosis (177, 183).
The overload of iodine in iodine- and Se-deficient rats does not mimic conditions leading to myxedematous cretinism. Thiocyanate overload instead of iodine might elicit the necrosis. It would aggravate the effects of iodine deficiency by competing with iodide for transport and generate toxic derivatives as well. Indeed, thiocyanate administration to iodine- and Se-deficient rats causes acute inflammation of the thyroid followed by extensive and prolonged fibrosis and atrophy of thyroid follicles.
The association of three factors, i.e., iodine and Se deficiencies plus thiocyanate overload, mimics in rats the phenotype of Central Africa myxedematous cretinism (177). Correction of the iodine and Se deficiencies appears the logical prevention strategy. Correcting the Se deficiency first would be a daring strategy, because it induces T4 deiodination and consequently increases loss of scarce iodine, which worsens the hypothyroidism and might lead to catastrophic thyroid failure (224).
C. Selenium deficiency and neurological cretinism
All three iodothyronine deiodinases are selenoenzymes, and Se deficiency decreases the type I and II enzyme activities by two different mechanisms (see Section VI). Type II and III deiodinases appear more resistant to Se deficiency. The low relative incidence of neurological cretinism in Africa might result from Se deficiency; low T4 deiodination in the mother and in the embryo would allow higher net T4 supply to the fetal brain, thereby mitigating at this level the decrease in maternal T4 due to iodine deficiency (166, 167, 180, 224). However, experiments in rats did not demonstrate higher T4 or T3 levels in fetal brains of Se-deficient mothers with iodine deficiency (225). Although this evidence does not exclude the postulated mechanism in humans, it certainly does not support it.
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VI. Selenoproteins and the Thyroid Axis |
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TopAbstractI. Historical AspectsII. Biosynthesis and Degradation...III. Recently Discovered...IV. Hormonal Regulation of...V. Selenium, Cell Defense,...VI. Selenoproteins and the...VII. Selenium and the...I. Selenium, the hormonal...J. Selenoproteins and the...References |
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). The exact mechanism of these reactions and their possible physiological cofactors remain unknown. In vitro, strong dithiol reductants such as DTT or dithioerythreitol (DTE) act as cosubstrates in this ping-pong sequential two-substrate reaction releasing free iodide from the enzyme intermediate or iodothyronine substrate. Three enzymes catalyzing iodothyronine deiodination have been identified, which differ in their substrate preference, reaction mechanism, inhibitor sensitivity, tissue- and development-specific expression, and regulation by their substrates or products as well as by other physiological factors and susceptibility to pharmacological agents (226, 227, 228).
1. The selenoprotein D1.
Type I iodothyronine D1 is the most abundant and best characterized of the three deiodinases (Table 3). D1 was established as a selenoprotein by a combination of metabolic in vivo labeling of the protein in Se-deficient rats with 75-selenite and concomitant in vitro affinity labeling of its active site with N-bromoacetyl derivatives of thyroid hormones (11, 12). These studies revealed the 27-kDa substrate binding subunit of D1 (229), which only functions as intact homodimer (230, 231), and established that one Se atom was present in the 27-kDa subunit active site (11). The tissue distribution and regulation of this 27-kDa subunit parallels that of the D1 activity in the rat, various cell lines, and other species. The cloning of the cDNA encoding the D1 27-kDa subunit and the identification of an in-frame UGA codon and a 3'-utr SECIS sequence, essential for translation of a functional D1 enzyme, confirmed the selenoprotein nature of D1 and later of other eukaryotic selenoproteins (13, 60). Subsequently, D1 genes have been cloned, and their products have been characterized in many eukaryotic species (232, 233). The 17.5-kB gene of the human D1 has been mapped to chromosome 1p32–33 and contains four exons (234). So far, no splice variants, mutants, or human D1 gene defects have been reported. However, polymorphisms in the 3'-utr or the human D1 gene have been associated with altered thyroid hormone and IGF-I serum levels (235, 236). Several kilobases of the promoter of the human gene have been cloned and partially characterized (237, 238, 239, 240). A series of putative regulatory consensus elements were postulated, and two complex thyroid hormone and retinoid-responsive elements have been functionally characterized in the human D1 promoter (63, 237, 238, 239). These comprise a direct repeat of three consensus half-sites (DR4 + 2) with a spacer of four bases conferring T3-response (DR4) and a spacer of two bases mediating retinoid response (DR2). A further combined T3 and retinoid-responsive direct repeat element with a spacing of 12 bases (DR12) is located downstream in the proximal promoter. Both elements act in tissue-specific context with respect to T3 and/or retinoid regulation of D1 reporter gene constructs. These elements can explain T3 stimulation of D1 expression in many tissues and nontransformed cell lines as well as retinoid induction of D1 in tumor cells (see Section VI.A.1).
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D1 catalyzes the 5'-deiodination of L-T4, rT3, and other iodothyronines (Fig. 7) or their sulfoconjugates. D1 also removes iodide from the 5(3) position of the tyrosyl ring at alkaline pH (249). Liver and thyroid D1 are assumed to produce most of the circulating T3 under normal conditions. D1 also participates in the local production of T3 from T4 in some organs. However, the extent is difficult to determine because many tissues express specific T3-carrier or transport systems as well as D2 (250, 251, 252, 253).
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Many hormonal, nutritional, and developmental factors modulate the expression and activity of D1 (226, 227, 259, 260). The substrate and/or products of the enzyme (T4, T3, 3,3-T2) induce its expression, whereas hypothyroidism decreases its activity in most tissues (226, 227, 261, 262, 263, 264, 265, 266, 267). In the thyroid, TSH and its cAMP-protein kinase A-signaling cascade increase D1 activity in several species (268, 269, 270). Se supply might affect this regulation because TSH enhances D1 mRNA abundance in Se-deficient rats but decreases it in Se-adequate conditions in FRTL-5 cells (269). Sex steroids exert tissue-specific effects on D1 expression. Although hepatic D1 is induced by testosterone, D1 activity is higher in pituitaries of female rats (271, 272, 273). Corticosteroid regulation of D1 expression and activity depends on the system and model investigated. Whereas most in vivo animal experiments reveal inhibition of D1 activity, dexamethasone stabilizes D1 mRNA and enhances T3 stimulation of D1 enzyme activity in some in vitro models (264, 265).
D1 activity is also increased by stimulation of the GH-IGF-I axis in most species and models analyzed (274, 275, 276, 277, 278, 279, 280, 281, 282, 283). It is not yet clear whether GH has a direct stimulatory effect independent of IGF-I. Increased serum T3/T4 ratio is interpreted as stimulation of hepatic D1 by GH. GH/IGF-I and their binding proteins also interfere with the Se homeostasis, and conversely, growth curves of Se-deficient animals are affected (78, 284). However, growth can be restored in Se-deficient rats by injections of 1 µg Se/100 g body weight, too low to normalize serum thyroid hormone levels, and the infusion of T3 alone does not increase the growth rate (284).
Fasting decreases and carbohydrate feeding markedly stimulates hepatic D1 activity, but the exact mechanisms involved remain elusive. In diabetic rats, expression of hepatic D1 is reduced but can be restored by T3 or insulin administration (285). Proinflammatory cytokines down-regulate D1 in liver and thyroid and up-regulate it in liver and pituitary (267).
Severe Se deficiency reduces D1 protein and activity in a tissue-specific manner, and repletion increases it (97) by combinations of mechanisms involving both D1 mRNA steady-state levels and posttranscriptional regulation (286, 287). Systematic analysis of modulation of the tissue-specific expression of various Sec-containing enzymes and proteins revealed a pronounced hierarchy in Se responsiveness and Se supply to individual selenoproteins in tissue-specific manner (98, 269, 288). In general, D1, an enzyme of low abundance, seems to hold a high rank in this hierarchy, at least above cGPx, enabling local and systemic production of T3 from T4 even at low available Se concentrations (97). In a cell culture model, D1 may even recruit Se liberated from the turnover of the more abundant selenoprotein cGPx for incorporation into newly synthesized D1 (97). Several tissues exhibit a further hierarchy. Whereas liver, kidney, heart, skin, and muscle are rapidly depleted from Se during severe deficiency, the thyroid, several other endocrine organs, the reproductive system, and the brain retain Se to a remarkable extent. In adult Se-deficient animals, PHGPx and even more D1 activity are kept at high levels in the thyroid (98).
Stabilized organoselenenyl iodides were used to mimic the Sec-containing active site of D1 and its reaction mechanism as enzyme-mimetic substrates. Propylthiouracil (PTU) reacts with the oxidized E-SeI enzyme intermediate, but not the native enzyme. Basic residues in the active site, such as the proposed histidine, which can form a selenenolate-imidazolium ion-pair (242), kinetically activate the SeI bond. Hydrogen-iodide-catalyzed disproportionation of E-SeI intermediates to diselenides may occur if sterically feasible in the enzyme. An E-SeI reaction with a selenol is much faster than with a thiol, and these factors might account for insensitivity toward PTU inhibition of D2 and D3 (289, 290, 291). PTU is inactive toward diselenides.
2. Type II 5'-deiodinase—a second selenoprotein involved in deiodination of T4 to T3.
The D2, like the D1, enzyme generates T3 from the prohormone T4 (Table 3). D2 has a higher affinity for T4 than D1 (KMapp = 2 nM T4), and shows high specificity for T4. Furthermore, D2 is rapidly inactivated by its substrate T4, but also by rT3 (292). Its transcription is inhibited by T3; thus, regulation is inverse to that of D1 and D3 (293). Tissue distribution, developmental profile, and regulation by hormones and other signals are distinct from that of D1 (294, 295). Therefore, D2 is assumed to generate T3 from local T4 sources for intracellular demands independent from circulating T3, and the contribution of D2 to circulating T3 is considered to be limited. The latter assumption has been thrown into question by the findings of significant mRNA and enzyme levels in the human thyroid and muscle and cells derived therefrom (296, 297). D2 activity was found in neonatal rat thyroid, but not in adult rat thyroid (294), and mRNA levels do not in all instances reflect expression and activity of the enzyme (298, 299, 300, 301).
In vitro determination of D2 activity takes advantage of its weak inhibition and the strong inhibition of D1 by the PTU drug. The mechanism of D2 reaction proceeds via a sequential two-substrate reaction without intermediate formation of an oxidized selenenyl residue (Fig. 2). D2 is thought to be unaffected by PTU, which forms a covalent intermediate with the oxidized selenenyl residue of D1 and reacts in a two-substrate ping-pong mechanism with formation of an oxidized enzyme intermediate (289, 290, 291).
The selenoprotein nature of D2 has been questioned, because several models have found no clear Se-dependent expression of D2 (287, 302). The identification of a functional SECIS element in the 3'-utr of the long D2 mRNA has only recently been achieved. Cloning of highly conserved orthologs to the D2 transcript, identification of full length cDNAs, characterization of the human D2 gene on chromosome 14q24.2–3 (303, 304, 305, 306, 307), and several in vivo and in vitro findings suggest that the D2 transcript encodes a functional D2 enzyme with a mass of 200 kDa (308). Strong experimental evidence for the selenoprotein nature of D2 encoded by the SECIS-containing D2 transcript was provided by experiments with a human mesothelioma cell line. High levels of expression of D2 transcripts, Se-dependent functional activity of D2, and 75Se-labeling of a p31 subunit were found (309). One study compared the expression and location of the D2 selenoenzyme transcript and the transcript of the cAMP-responsive p29 nonseleno T4 binding subunit (300). A different location of the two transcripts was reported in the rat brain; p29 was expressed in neurons and in all the regions of the blood-cerebrospinal fluid barrier, but in different cell types than the D2 selenoprotein transcript, with the exception of the tanycytes. This does not support the assumption that p29 has a functional relationship with D2 (310).
Whereas the human D2 gene encodes for two SeCys residues in the protein, in most other species only one highly conserved active-site SeCys residue is found. The second SeCys residue 266 in the human D2, located seven codons upstream of the stop codon, is not essential for enzyme function. Site-directed mutagenesis to a cysteine residue or a stop codon had no effect on enzyme activity but modified Se incorporation (311).
D2 is highly expressed in the central nervous system (CNS), with the highest levels in astroglial cells and tanycytes. Neurons, in which most of the T3 receptors are expressed, show rather low D2-enzyme activity. D2 transcripts have also been localized to tanycytes (312, 313, 314). Thus, D2, locally generating the active hormone T3 from its precursor T4, and the nuclear T3 receptors, mediating most of thyroid hormone action, are localized within different cell types. This suggests a regulated efflux and transport of T3 from its intracellular site of production in glial cells to surrounding neurons containing T3 receptors (253). Cell-specific membrane transporters such as MCT-8 (315) and OATP14 (316) might independently control influx and efflux of T4, T3, and their metabolites. Both T4 and rT3 but not T3 are potent regulators of D2 inactivation (317). Nonnuclear receptor-mediated mechanisms of thyroid hormone action might also play an important role in hormone action (318). Thyroid hormones also regulate neuronal migration and neurite outgrowth as well as laminin expression in rat astrocytes and within the rat cerebellum (319, 320). Because laminin is produced and secreted by astrocytes, which have low numbers of thyroid hormone receptors but high D2 activity, thyroid hormone-dependent alteration of laminin secretion might be mediated by an extranuclear thyroid hormone effect independent of T3 receptors.
In the hypothalamus, in situ hybridization in combination with immunohistochemistry for the glial cell marker glial fibrillary acidic protein revealed a colocalization of D2 transcripts in glial cells of the median eminence and the arcuate nucleus, but not the paraventricular nucleus. This indicates a close relationship between local thyroid hormone production in the hypothalamus and neuroendocrine TRH-producing cells in the paraventricular nucleus (321).
In the hypothyroid rat brain, D2 transcripts were found elevated in relay nuclei and cortical targets of the primary sensory and auditory pathways (322). The occurrence of D2 transcripts in the cochlea of the developing rat suggests a major function of locally formed T3 in this structure (323). Thyroid hormone receptor (TR) is expressed in the sensory epithelium, whereas D2 is found in the periostal connective tissue, which might thereby control T4 deiodination and T3 release for action in the epithelium in a paracrine manner.
cAMP stimulation of D2 activity and expression has been demonstrated in glial cells, human thyroid, and brown adipose tissue of rodents (309, 324, 325, 326). In brown adipose cells, D2 is highly expressed and generates T3 essential for stimulation of expression of uncoupling proteins and thermogenesis in synergism with catecholamines. A cAMP-responsive element has also been identified in the human D2 gene and functionally characterized in thyrocytes (327, 328). In rat astrocytes, cAMP stimulation of D2 activity has been linked to the recruitment of a 60-kDa cAMP-dependent protein to the p29 catalytic subunit affinity-labeled by BrAcT4 to yield the 200-kDa holoenzyme complex. During this cAMP-dependent stimulation of D2 activity, its p29 subunit is translocated from the perinuclear space to the inner leaflet of the plasma membrane coincident with appearance of deiodinating activity (325). The promoter of the human, but not the rat, D2 gene contains a functional TTF-1 response element (329). Stimulation of glial cell D2 by nicotine and its inhibition by mecamylamine, which blocks nicotine binding to nicotinic acetylcholine receptors, could influence brain function via modulation of local T3 production (330).
A study reports a transgenic mouse model in which an artificial gene construct comprising the coding region of the human D2 and the rat SePP SECIS element flanked by the human GH polyadenylation signal was expressed under the control of exons I and II of the mouse -myosin heavy chain gene promoter (331, 332). These mice had elevated cardiac D2 activity, unchanged cardiac T3 levels, and unaltered plasma hormone levels and growth rate. Nevertheless, signs of cardiac hyperthyroidism were observed associated with increased adrenergic responsiveness. Conversely, targeted deletion of D2 in mice revealed a mild phenotype (increased serum levels of T4 and TSH, but normal T3), mild growth retardation in males, and impaired cold adaption (333, 334). This indicates either functional redundancies among the deiodinases or efficient adaption of the components of the thyroid hormone network to failure of a component.
The novel identification of mutations in the human SBP2 gene (22), which led to a phenotype of the thyroid hormone axis resembling that of D2 knockout mice (333), illustrates the importance of Se in thyroid hormone economy and especially for adequate function of D2. Elevated serum TSH, T4, rT3, and low T3 are accompanied by low serum levels of Se, GPx, and SePP, indicating a major disturbance of Se homeostasis but an early manifestation of this genetic defect in the thyroid hormone axis.
In animal models of Se deficiency, only minor alterations of Se content are observed in most endocrine organs and in the CNS. Similarly, only minor alterations of D1, D2, and D3 activity were found in the CNS under Se depletion and repletion (335). The major regulator of D2 expression in the brain is the thyroid hormone status itself. In hypothyroidism, D2 mRNA is increased severalfold in glial cells and in interneurons in the regions related to primary somatosensory and auditory pathways (322). Because the brain strongly depends on T4 supply from the thyroid and circulating serum T3 probably reaches the brain only in limited quantities or under pathological conditions, proper thyroid function, and hence adequate Se supply, is crucial both during development and in the adult organism. Apart from thyroid hormone, stress, circadian rhythm, and several neuroactive drugs affect brain deiodinase enzymes and local thyroid hormone levels strongly (336, 337, 338, 339).
Se deficiency is known to impair cold tolerance in animals, which might be related to lower expression of D2 in brown adipose tissue associated with decreased T3 production and subsequent reduction of uncoupling protein expression and catecholamine-stimulated thermogenesis (340). A rat astrocyte culture model has shown that the Se status modulates cAMP stimulation of D2 expression (302).
3. Type III 5-deiodinase—the selenoprotein catalyzing T4 and T3 inactivation.
The selenoenzyme D3 inactivates thyroid hormones, both the prohormone T4 and its active metabolites such as T3 or 3,5-T2. D3 does not metabolize T4-sulfate and T3-sulfate (341). The products of deiodination of iodothyronines at the tyrosyl ring in 5-(or 3-) position (Fig. 3) are devoid of thyromimetic activity and do not bind to nuclear T3 receptors. The main metabolite of D3, rT3, competes for T4 deiodination by D1 and thus might have a regulatory function in thyroid hormone metabolism. Because circulating rT3 levels are in the range of T3 and high rT3 formation is found in the CNS (342), a biological role for this metabolite during brain development, such as
