Biochemical Mechanisms of Thyroid Hormone Deiodination

THYROID Volume 15, Number 8, 2005 © Mary Ann Liebert, Inc.

George G.J.M. Kuiper, Monique H.A. Kester, Robin P. Peeters, and Theo J. Visser

Deiodination is the foremost pathway of thyroid hormone metabolism not only in quantitative terms but also because thyroxine (T4) is activated by outer ring deiodination (ORD) to 3,3’,5-triiodothyronine (T3), whereas both T4 and T3 are inactivated by inner ring deiodination (IRD) to 3,3’,5-triiodothyronine and 3,3’- diiodothyronine, respectively. These reactions are catalyzed by three iodothyronine deiodinases, D1-3. Although they are homologous selenoproteins, they differ in important respects such as catalysis of ORD and/or IRD, deiodination of sulfated iodothyronines, inhibition by the thyrostatic drug propylthiouracil, and regulation during fetal and neonatal development, by thyroid state, and during illness. In this review we will briefly discuss recent developments in these different areas. These have resulted in the emerging view that the biological activity of thyroid hormone is regulated locally by tissue-specific regulation of the different deiodinases.

HYROID HORMONE is essential for growth, development, thyrostatic drug 6-propyl-2-thiouracil (PTU). D1 activity is Tand regulation of energy metabolism (1–3). Amphibian positively regulated by T3, reflecting regulation of D1 ex- metamorphosis is an important example of thyroid hormone pression by T3 at the pretranslational level. actions on development (4). Equally well known is the crit- In humans, D2 activity is found in brain, anterior pitu- ical role of thyroid hormone in development and function itary, placenta, thyroid and skeletal muscle, and D2 mRNA of the human central nervous system (5,6). Thyroid hor- has also been detected in the human heart. In rodents D2 is mone is produced by the thyroid in the form of the biolog- also expressed in brown adipose tissue. D2 has only ORD ically inactive precursor thyroxine (T4). The prinicipal bioac- activity, preferring T4 over rT3 as the substrate, with appar- tive form of the hormone is triiodothyronine (T3). In ent Km values in the nanomolar range. In general, D2 activ- humans, only approximately 20% of T3 is secreted by the ity is increased in hypothyroidism and decreased in hyper- thyroid; most circulating T3 is derived from outer ring deio- thyroidism. Both pre- and posttranslational mechanisms are dination (ORD) of T4 in peripheral tissues. Both T4 and T3 involved in the regulation of D2 expression by thyroid state, undergo inner ring deiodination (IRD) to metabolites which with distinct roles for T3, and for T4 and rT3, respectively. do not interact with T3 receptors, reverse triiodothyronine Although perhaps D2 in skeletal muscle may contribute to (rT3) and 3,3 -diiodothyronine (3,3 -T2), respectively. Thus, circulating T3, the enzyme is particularly important for local ORD is regarded as an activating pathway and IRD as an T3 production in brain and anterior pituitary. inactivating pathway. ORD is also the main pathway for the In human and rodents, D3 is located in brain, placenta, metabolism of rT3, representing another route for the gen- pregnant uterus, and fetal tissues. D3 has only IRD activity, eration of 3,3 -T2. Three iodothyronine deiodinases are in- and is thus important for the inactivation of thyroid hor- volved in the deiodination of iodothyronines, namely, mone. It shows preference for T3 over T4 as the substrate, D1–D3 (7–9). with apparent Km values in the nanomolar range. The high In humans and rodents, D1 is located primarily in liver, D3 activity in placenta, pregnant uterus and different fetal kidney, and thyroid. Lower D1 activities are expressed in tissues seems to serve the purpose of protecting the fetus other tissues, including rat anterior pituitary. Although D1 against undue exposure to active thyroid hormone that may has both ORD and IRD activities, it appears particularly im- be detrimental for the development of different tissues, in portant for the generation of plasma T3 and clearance of particular the brain. In brain, D3 activity is increased in hy- plasma rT3. ORD of rT3 is the most efficient reaction cat- perthyroidism and decreased in hypothyroidism but the alyzed by D1, while IRD of both T4 and T3 are strongly ac- mechanism of this regulation remains to be established. celerated by sulfation of these iodothyronines. Michaelis In this short review we will focus on recent insights in Menten constant (Km) values for substrates of D1 are in the deiodinase structure-function relationships and physiologi- micromolar range. The enzyme is potently inhibited by the cal roles of deiodinases. For a more in-depth discussion on

Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands.

787 788 KUIPER ET AL. the concepts underlying much of the work to be presented Iodothyronine substrate interaction the reader is referred to earlier reviews (8–11). Cloning of D1 from various species (rat, mouse, cat, dog, human) and careful analysis of kinetic properties with a Structure-Function Relationship of range of substrates (T , T , rT , rT S, T , T S) has enabled the Iodothyronine Deiodinases 4 3 3 3 2 2 identification of a region that is involved in substrate inter- Catalytic center action. Comparative structural–functional analysis of human and dog D1 enzymes showed that the region between amino Iodothyronine deiodinases are selenoproteins, containing acid residue 30 and 70 accounts for the difference in K value a single selenocysteine residue (SeC) in the core catalytic cen- m for rT ORD between dog and human D1 (19). Dog D1 has ter. This core catalytic center consists of approximately 15 3 an approximately 30-fold higher K for rT ORD than hu- amino acid residues surrounding the SeC and is highly con- m 3 man D1. More detailed studies demonstrated that it is mainly served within and between the deiodinase subtypes. The SeC the Phe65Leu substitution that explains the slow ORD of rT is encoded by a UGA stop codon which in the presence of a 3 by dog D1 versus human and rat D1 (19). The same type of so-called SECIS (selenocysteine insertion sequence) element studies comparing cat and rat D1 enzyme also indicated that in the 3 untranslated region (UTR) is recoded from a stop the region between residue 40 and 70 is involved in substrate to a SeC codon (12). The SeC residue is essential for enzyme interaction. By site-directed mutagenesis it was found that a activity because replacement with Ala or Ser residues (es- combination of mutations was necessary to improve the sentially replacing the SeH group by H or OH) eliminates deiodination of rT by cat D1. For efficient rT deiodination, activity. As far as we know only replacement of SeC with 3 3 a Phe at position 65 and the insertion of the Thr-Gly-Met- Cys (substituting S for Se) in D1, D2, or D3 maintains enzy- Thr-Arg sequence (residue 48–52) as well as the amino acids matic activity, although with strongly reduced substrate Gly and Glu at position 45–46 are essential (20). An intrigu- turnover numbers and significantly increased K values for m ing property of cat D1 is the facilitated deiodination of rT S, the iodothyronine substrates (13–15). For some reason dur- 3 and the combination of the described changes did not affect ing evolution nature has decided that deiodinases should be this property (V /K rT 3 and V /K rT S 81). selenoenzymes, despite the fact that the synthesis of a se- max m 3 max m 3 The negatively charged sulfate group of rT S might interact lenoprotein is expensive for the cell. From an energetic point 3 with the positively charged side group of a basic amino acid of view the extraction of iodonium (I) from an aromatic ring (Lys, Arg), thereby stabilizing interaction with D1. Our stud- is a difficult step and most likely the more negatively charged ies indicated that this basic amino acid is probably situated selenol group (SeH ↔ Se) is better capable of accomplish- outside the region between residue 40 and 70, and remains ing this than the sulfydryl group (SH). The selenol group to be identified. The fact that D1 catalyzes ORD (rT3, rT3S, might interact with side groups of other amino acid residues T ) and IRD (T , T S, T , T S) suggests different orientations facilitating its deprotonation. For D1 the formation of an es- 4 4 4 3 3 of substrate binding within a single site, so that either the sential imidazolium–selenolate ion pair was postulated on iodines of the inner ring or of the outer ring are in close prox- the basis of experiments with histidine-directed reagents and imity of the catalytic center. In other words, the D1 substrate side-directed mutagenesis studies (8,16,17). binding site is very flexible and therefore it will be difficult More detailed insights in deiodinase structure and cat- to gain more insight in the structure–activity relationship by alytic mechanism must come from the three-dimensional site-directed mutagenesis studies with the thus far cloned D1 structure when this is resolved by crystallographic studies. enzymes, unless D1 variants from other species turn up Unfortunately, these studies are greatly hampered by the dif- which for instance lack IRD activity or do not show facili- ficulties encountered with overexpressing these membrane- tated deiodination of sulfated iodothyronines. For similar integrated enzymes in a soluble and active form. An alter- reasons, that is no large variations in kinetic properties for native strategy consists of attempting to model deiodinase different substrates among D2 and D3 enzymes in various structure on the basis of structural resemblance to other pro- species, no progress has been made in the identification of teins for which experimental structure information exists. substrate interacting regions in D2 or D3. Hydrophobic cluster analysis revealed that the extramem- brane portion of deiodinases belongs to the thioredoxin-fold PTU inhibition of D1 superfamily and that a region within this fold shared strong similarities with the active site of iduronidase (18). This low- A largely unsolved question concerns the basis for the po- resolution deiodinase structural model consists of a single tent inhibition of mammalian D1, but not teleost fish D1, by transmembrane domain and a hinge or linker domain con- PTU. The catalytic cycle of D1 appears to exist of two half nected to a globular domain containing the active center. The reactions: (1) transfer of an I from the substrate to the se- model provided novel information about conserved amino lenolate (Se) anion of SeC and (2) reduction of the selenenyl acid positions in the active center surrounding the core cat- iodide (SeI) generated by a thiol cofactor. PTU inhibition of alytic center with the SeC residue. These amino acid residues D1 is uncompetitive with iodothyronine substrate, and there are very well conserved between the deiodinase subtypes is strong evidence that PTU reacts with the SeI intermediate and it was shown that variations of specific amino acid (Fig. 1) (17,21). The selenenyl sulfide thus formed is consid- residues are linked to functional differences among D1, D2, ered to be a dead-end product because it does not react with and D3 (see below). The model involved part of the protein thiols under physiologic conditions. (from amino acid residue 75 up to the C-terminus) and did D1 from various teleost fish species as killifish (Fundulus not provide structural information with regard to the trans- heteroclitus) and tilapia (Oreochromis niloticus) have greatly re- membrane domain and adjacent region known to be induced sensitivity toward inhibition by PTU (IC50 1000 M volved in iodothyronine substrate interaction. for tilapia D1 versus 1 M for human D1). Also, sensitivity THYROID HORMONE DEIODINATION 789

differ with respect to tissue distribution, ontogenic profiles and other aspects such as substrate preference and inhibitor profile. Thus far, it was thought that iodothyronine deiodinases are vertebrate innovations, but the discovery of iodothyronines, homologues of iodothyronine deiodinases, and thyroid hormone receptors (TR) in invertebrates has shed new light on the coevolution of proteins involved in thyroid hormone synthesis, iodothyronine deiodinases and TRs (24–27). Ascidians or sea squirts (for instance Ciona and Halocyn- thia) belong to the phylum Urochordata. They are often re- FIG. 1. Putative catalytic mechanism of type I deiodinase, ferred to as protochordates because during the larval stage and its inhibition by propylthiouracil (PTU), iodoacetate they possess chordate characteristics, most notably the tail (IAC), and gold thioglucose (GTG). contains a notochord and a dorsal hollow nerve cord. After a free-swimming stage, the simple tadpole-like larvae attach to a substrate and undergo metamorphosis that includes tail for the other D1 inhibitors goldthioglucose (GTG) and iodoac- loss and rearrangement of the internal organs. Subsequently, etate (IAc) is reduced (22,23). Kinetic data suggest that GTG in the adult form, the similarities to chordates are lost. Al- and IAc react with the SeC residue in its reduced (SeH) form though no clear role for thyroid hormones in adult ascidians (25) (Fig. 1). We have recently characterized a Xenopus laevis has been established, there are studies suggesting a role for D1 enzyme (K T and rT 0.2 M) which is not inhibited m 4 3 T in the metamorphosis from the larval to the adult stage by PTU and also displays reduced sensitivity for GTG and IAc 4 (28). Indeed, the first evidence of an organ related to the ver- (unpublished data). The PTU insensivity of teleost fish and tebrate thyroid gland is found in protochordates. The ascid- ampibian D1 proteins might be related to a different catalytic ian endostyle is a mucus-secreting pharyngeal organ that mechanism not involving the formation of a SeI intermediate. facilitates filter feeding. The endostyle has iodine-concen- However, that would not explain the reduced sensitivity to- trating activity, and the biosynthesis of thyroid hormone by ward GTG and IAc. More likely, the reduced sensitivity for this organ is well documented (25,26). An endostyle is also GTG, IAc, and PTU reflects a decreased reactivity of the se- found in cephalochordates (Amphioxus) and in larval lam- lenol group of the SeC residue as such. The substrate turn- prey, where it transforms into a follicular thyroid gland dur- over number of tilapia D1, and probably also the other PTU- ing metamorphosis (25). insensitive D1 enzymes, is strongly reduced in comparison to We have recently characterized an iodothyronine deiod- rat and human D1 (22). The selenol group may interact with inase from Halocyntha roretzi (hrDx) as a high-K enzyme side chains of other amino acid residues, stimulating depro- m with ORD activity toward T and rT . This enzyme shows tonation at physiologic pH. In this regard it is interesting to 4 3 a temperature optimum between 20°C and 30°C and has ki- note that the pI value of PTU-sensitive D1 enzymes is 9–10, netic characteristics resembling vertebrate D1 and D2 (29). whereas it is 6–7 for PTU insensitive D1 enzymes. At various The 3-UTR of the hrDx mRNA contains a SECIS element as positions in PTU-sensitive D1 enzymes basic or neutral amino revealed with the SECISearch program (12). The existence acid residues are present were PTU-insensitive D1 enzymes of an ascidian homologue of vertebrate deiodinases raises have acidic or neutral amino acid residues. Whether these dif- the hypothesis that also in protochordates the prohormone ferences are related to D1 substrate turnover numbers and in- T is activated by ORD to T . Examination of the deduced hibitor profiles remains to be investigated. Apart from a pu- 4 3 amino acid sequence of hrDx shows that the overall ho- tative role for residues outside the core catalytic center there mology of hrDx with other deiodinase proteins is not high is experimental evidence that residues in the immediate vicin- (approximately 30%). Approximately the same level of ho- ity of the SeC residue play a role in determining PTU sensi- mology is found if one compares any one of the vertebrate tivity. Within the core catalytic center surrounding the SeC deiodinases to any of the other deiodinases. The catalytic residue the PTU-sensitive D1 enzymes all share the sequence center around the SeC residue is completely conserved, as Gly-Ser-Cys-Thr-SeC-Pro-Ser-Phe, while the PTU-insensitive it is in all deiodinase cDNA sequences known to date (Fig. D1 enzymes share the sequence Gly-Ser-Cys-Thr-SeC-Pro- 2). The availability (24) of genomic and EST sequences of Pro-Phe. So, the residue 2 positions downstream from SeC is two other ascidian species, Ciona intestinalis and Ciona savi- Pro in PTU-insentive D1 enzymes and Ser in PTU-sensitive gnyi, enabled us to search for homologues of the hrDx in D1 enzymes. As expected, the Ser to Pro mutation in the PTU- these sea squirts. We have identified two sequences from sensitive human D1 enzyme (Ser128Pro) did decrease PTU each species that have high homology with hrDx, namely, sensitivity, but the reverse mutation in the PTU insensitive ciDx and ciDy for C. intestinalis and csDx and CsDy for C. tilapia D1 enzyme (Pro128Ser) did not restore PTU sensitiv- savignyi. The existence of two different deiodinase-like se- ity (18,22). In conclusion, amino acid residues in the immedi- quences in the Ciona species is particularly interesting since ate vicinity of the SeC residue influence PTU sensitivity, but it might indicate that ascidians possess not only a D1/D2- there is also an important modifying role for residues in other like deiodinase but also a D3-like deiodinase. Using the data segments of the D1 enzyme that remain to be elucidated. for deiodinase sequences from vertebrate and invertebrate species it was possible to construct a phylogenetic tree of Invertebrate deiodinases the deiodinases (Fig. 3). The tree resolved into four main All vertebrates (mammals, birds, teleost fish) have been clusters containing the three vertebrate deiodinase sub- shown to express D1, D2, and D3 activity, although they may types, with the invertebrate deiodinases forming a seperate 790 KUIPER ET AL.

FIG. 2. Comparison of the deduced amino acid sequence of the Halocyntha roretzi (hrDx) protein with those of human (hsD1), chicken (ggD1), and tilapia (onD1) type I deiodinases; human (hsD2), chicken (ggD2), and killifish (fhD2) type II deiodinases; and human (hsD3), chicken (ggD3), and tilapia (onD3a) type III deiodinases. The SeC residue is denoted by U (residue 133 of hrDx).

cluster, branching close to the origin of the tree. All in all Indeed, all iodothyronine deiodinases catalyze the deiod- there is now sound evidence that the iodothyronine deiodination of iodothyronines only in the presence of thiol-con- inases as well as TRs arose in a urochordate, or perhaps even taining compounds such as dithiothreitol (DTT). However, earlier in evolution since iodothyronines have effects on it should be mentioned that for none of the deiodinases the echinoderms (27,30) such as sea urchins and sand dollars. natural cofactor has been identified with certainty, although This indicates that iodothyronines and possibly TRs and stimulation of D1 activity has been demonstrated with the deiodinases arose earlier in evolution than receptors for gen- glutaredoxin system, the thioredoxin system, and dihy- der/adrenal steroids and steroid-synthesizing enzymes. drolipoamide (33–37). In addition, the stochiometry of the Steroid and vitamin D receptors arose in cephalochordates deiodination reaction has not been settled in terms of the ox- (for instance Amphioxus), or jawless fish such as Lamprey idation of thiol cofactor, however, obvious this may appear (31,32). from the above equation. In view of the high degree of homology between the dif- Biochemistry of Deiodination ferent deiodinases, it is logical to assume that they deiodi- nate their substrates by similar biochemical mechanisms. Be- Enzymatic deiodination of iodothyronines is a reductive cause all iodothyronine deiodinases characterized so far process. This is obvious considering the substrates and prod- have been identified as selenoproteins containing a reactive ucts of the overall iodothyronine deiodination reaction: SeC residue in corresponding positions of the amino acid se- TIn 2RSH TIn-1H RSSR H I quences, it is also reasonable to ascribe a special function to THYROID HORMONE DEIODINATION 791

both T4 and T3 by D1, whereas ORD may either be stimu- lated (3,3 -T2), unaffected (rT3) or even inhibited (T4) if these compounds are sulfated (38). As far as this has been tested, D2 and D3 are incapable of deiodinating sulfated substrates. Also the catalytic mechanisms appear to differ between the different iodothyronine deiodinases. For instance, D1 show ping-pong type enzyme kinetics, characterized by par- allel lines in the Lineweaver-Burk plot of deiodination rate versus substrate concentration at different fixed cofactor concentrations (39). This is suggestive for the alternate reaction of substrate and cofactor with different enzyme forms (the native enzyme and an enzyme intermediate generated during the catalytic cycle). In contrast, both D2 and D3 show sequential type reaction kinetics, characterized by converging lines in the Lineweaver-Burk plots at varying DTT concentration (39). This suggests the formation of ternary enzyme- substrate-cofactor complexes during D2 and D3-catalyzed deiodinations. Perhaps the most well-known distinction between the deiodinases is the potent inhibition of D1 by PTU in contrast to the lack of inhibition of D2 and D3 by PTU. This inhibi- FIG. 3. Phylogenetic tree of the known, complete deduced tion of D1 by PTU is uncompetitive with substrate and com- amino acid sequences of the vertebrate deiodinases, and the petitive with cofactor (DTT). These findings suggest that Halocyntha rovetzi (hrDx) deiodinase homolog, prepared with PTU and DTT react with the same enzyme intermediate gen- the Clustal W program package. cc, Cyprinus carpio; cf, Canus erated from D1 during deiodination, most probably the se- familiaris; ci, Ciona intestinalis; cs, Ciona savignyi; dr, Danio re- lenenyl iodide (SeI) group (see above and Fig. 1). The lack rio; fc, Felis catus; fh, Fundulus heteroclitus; gg, Gallus gallus; hr, Halocynthia roretzi; hs, Homo sapiens; mm, Mus musculus; of PTU inhibition of D2 and D3 would then imply that such nf, Neoceratodus forsteri; oa, Ovis aries; ol, Oryzias latipes; om, an SeI intermediate is not generated during deiodination cat- Oncorhynchus mykiss; on, Oreochromis niloticus; rc, Rana cates- alyzed by these enzymes. biana; rn, Rattus norvegicus; sa, Sparus aurata; sm, Suncus mur- However, recent findings cast doubt on the hypothesis inus; ss, Sus scrofa; tn, Tetraodon nigroviridis; tr, Takifugu that the catalytic mechanisms of the deiodinases are princi- rubripes; xl, Xenopus laevis. ply different. The dogma that D1 is inhibited by PTU has been contradicted by characterization of D1 from different fish species and from X. laevis that are poorly inhibited by the selenium in the deiodination process (9). This is sup- PTU. This has been related to the amino acid two positions ported by observations of marked decreases in enzyme ac- downstream of Sec, namely, Ser in all PTU-sensitive D1 vari- tivity after replacement of Sec by Cys, or even a complete in- ant, Pro in all PTU-insensitive D1, D2, and D3 enzymes (see activation of the deiodinases if Sec is replaced by Ser, Ala, above). Site-directed mutagenesis of Pro to Ser was shown or other amino acids (see above). to turn human D2 into a PTU-sensitive enzyme. In addition, Despite the high similarities between the structures of the it changed the kinetic mechanism of human D2 from se- deiodinases and the reactions they catalyze, there are also quential into ping-pong–type (18). However, substitution of obvious differences in their catalytic properties (Table 1). Ser for Pro in tilapia D1 did not increase the PTU sensitiv- Thus, D2 catalyzes only ORD, and D3 catalyzes only IRD, ity of this enzyme although it did change the ratio of ORD whereas D1 has both ORD and IRD activities (9). Remark- versus IRD catalysis (22). This may be explained by addi- able differences in substrate specificity are also noted in par- tional alterations in the enzyme active center impacting on ticular if the behavior of sulfated iodothyronine derivatives its catalytic potential, that is, the Asn to Ser mutation 6 po- are considered. Sulfation markedly accelerates the IRD of sitions upstream of the Sec residue (unplished observations).

TABLE 1. CHARACTERISTICS OF THE THREE IODOTHYRONINE DEIODINASES

D1 D2 D3

Deiodination ORDIRD ORD IRD Preferred substrates rT3 T4, T3 T4 rT3 T3 T4 Sulfation of substrates Stimulation Inhibition Inhibition Kinetic mechanism Ping-pong Sequential Sequential a Inhibitors (IC50, M) Propylthiouracil (PTU) 10.02 1000 1000 Iodoacetate (IAc) 1.02 1000 1000 Goldthioglucose (GTG) 0.02 1000 1000

aData derived from Wassen et al.7 ORD, outer ring deiodination; IRD, inner ring deiodination; rT3, reverse triiodothyronine; T4, thyroxine; T3, triiodothyronine 792 KUIPER ET AL.

Although it remains to be determined if the lack of the hy- port this rate-limiting role of T3 transport across the plasma droxyl group of Ser or the structural change associated with membrane for subsequent T3 metabolism (unpublished ob- its replacement by Pro is the actual cause of the loss of PTU servations). This is difficult to envisage in a situation where inhibition, it seems unlikely that the catalytic mechanism of the D3 active center is exposed on the cell surface. Therefore, the deiodinases is principly altered by the Ser-Pro inter- more studies are required to establish the exact subcellular change. Therefore, it is also unlikely that the formation of a location of D3. SeI intermediate is what distinguishes the catalytic mecha- Recently the gene coding for iodotyrosine dehalogenase nism of D1 from that of D2 and D3. (DEHAL1) has been characterized in humans and other The generation of a SeI intermediate during the catalytic species (44,45). DEHAL1 is a member of the nitroreductase cycle of D1, and perhaps also that of D2 and D3, implies that family, is expressed in the thyroid, liver and kidney, and uses enzymatic deiodination represents an electrophilic substitu- NADPH as the cofactor and FMN (flavin mononucleotide) tion of iodine by hydrogen. This means that iodine leaves as a prosthetic group for the deiodination of iodotyrosines. the substrate as an iodonium (I) ion, which would be as- It seems likely that information about the biochemical mech- sisted by the selenolate (Se) anion of Sec under formation anism of catalysis by DEHAL1 may be useful for a better un- of the SeI enzyme intermediate. However, it should be derstanding of the mechanism of deiodination by iodothy- stressed that the actual formation of this SeI intermediate has ronine deiodinases. not been demonstrated. This would also be a tough task, considering the instability of such a group. Also the putative co- Regulation of Deiodinases During valent complex resulting form the reaction of PTU with the Human Development SeI intermediate has not been identified, which would be less D3 in placenta and uterus of a problem, as it is thought to be a much more stable adduct. D3 plays an essential role in the regulation of the fetal The nucleophilic substitution of I by H in the outer ring is thyroid hormone status. It is highly expressed in fetal tis- promoted by the electron-donating effect of the (dissociated) sues such as the liver (46), placenta (47–50), and pregnant phenolic hydroxyl group. It is more difficult to envisage nu- uterus (51). By immunohistochemistry, human placental D3 cleophilic substitution of the outer ring iodines if this OH was shown to be located in syncytiotrophoblasts and cy- group is sulfated, or of the iodines in the inner ring which totrophoblasts (49,50), in the fetal endothelium of chorionic lacks an electron-donating OH group. It is possible that the villi, and in the maternal decidua (50). Compared to the nucleophilic substitution of iodine by hydrogen is facilitated adult, plasma T3 concentrations are very low in the fetus, by prior nucleophilic addition of a thiolate (S ) or the se- whereas levels of rT3 are more than 10-fold higher than in lenolate (Se ) group to the 2/6 or 2/6 position (i.e., adja- the adult (52). This fetal profile has been explained by the cent to the carbon carrying the I substituent). Such a mech- high D3 activities in the placenta, which limits transpla- anism has also been proposed for the dehalogenation of cental passage of maternal T4 and T3 to the fetus by con- iodinated and brominated uridine derivatives by thymidy- verting T4 to rT3 and T3 to 3,3 -T2 during placental transfer late synthetase (40). Such a mechanism could actually be (53,54). tested by analyzing the effects of substituents in the 2/6 In addition to the placenta, the uterus may also play a role and/or 2/6 positions on the deiodinaton of iodothyronine in the regulation of thyroid hormone bioactivity during fe- derivatives. tal development. It has been demonstrated that in the rat Although the different deiodinases are integral membrane pregnant uterus over a period of 48 to 72 hours after im- proteins located in the endoplasmic reticulum or in the plantation the expression of D3 is induced more than 200- plasma membrane, it is likely that they have their active cen- fold, to levels higher than in any other tissue (51,55). This in- ters oriented towards the cytoplasm. This is also the only duction of D3 seems to be a consequence of the implantation compartment with a reductive environment containing high process, since a similar increase of D3 expression is also (up to 10 mM) concentrations of reduced glutathione, the found in artificially decidualized uterine horns, but not in most abundant intracellular thiol compound (35). This re- undecidualized uterine horns from pseudopregnant rats ductive environment is essential to sustain reductive deiod- (55). Furthermore, in ovariectomized rats, Wasco et al. (55) ination by the deiodinases. In a recent study carried out demonstrated that estradiol and progesterone act synergis- largely with cells transfected with the different types of deio- tically to increase uterine D3 activity, which indicates that dinases, evidence was obtained that D1 is localized in the steroid hormones are likely to contribute to the regulation of plasma membrane with the largest part of the protein, in- uterine D3 expression. Immunolocalization studies have cluding the active center, exposed to the cytoplasm. D3 was shown that initially (at embryonic day 9 [E9]) D3 is localized also localized in the plasma membrane but it was found to at the implantation site in uterine decidual tissue and later have an opposite orientation, that is, with its active site ex- (E12 and E13) it is localized in the single cell layer of the ep- posed on the extracellular cell surface (41). Evidence was also ithelium lining the uterine lumen (51). obtained for rapid cycling of D3 between the plasma mem- In addition to the placenta and the uterus, D3 has also brane and endosomes. These findings are in contrast to ex- been demonstrated to be expressed at other maternal–fetal pectation since the extracellular, oxidative environment interfaces, such as in skin, in umbilical arteries and vein, and would not favor reductive deiodination by D3. Furthermore, in the fetal respiratory, digestive and urinary tract epithe- indirect evidence in patients with mutations in the MCT8 T3 lium (50). Therefore, together with the placenta, the preg- transporter suggest that T3 transport into the cell is required nant uterus and possibly also other sites of the maternal–fe- for deiodination by D3 (42,43). Recent studies in our labora- tal interface may serve to limit the exposure of the tory involving cotransfection of cells with MCT8 and D3 sup- developing fetus to maternal thyroid hormone. THYROID HORMONE DEIODINATION 793

D1 and D3 in fetal liver

The high rT3 versus low T3 in fetal serum has long been considered to be the result of a combination of the high D3 activity in the placenta and a low hepatic D1 activity. Indeed, hepatic D1 activity starts to be expressed in late stages in the rat, while hepatic D3 expression is low at all stages of rat development (56,57). However, our study of the ontogeny of hepatic D1 and D3 during human fetal development showed that substantial D1 activity (i.e. approximately 25% of that in the normal adult) is already expressed in the human fetal liver after the first trimester, and that D3 activities are high during fetal development, declining after midgestation to lower levels around term (46). Therefore, in addition to the high D3 activity in the utero–placenta unit it is rather the high hepatic D3 activity than a low hepatic D1 activity that contributes to the regulation of fetal circulating T3 levels. In agreement with this, studies in the embryonic chicken have clearly established a negative correlation between liver D3 expression and serum T3 levels (1,58). FIG. 4. Mean ( standard error [SE]) D3 activities and reverse triiodothyronine/thyroxine (rT3/T4) ratios in different D2 and D3 in fetal brain and local T3 levels human fetal brain regions. CC, cerebral cortex; CP, choroid plexus; GE, germinal eminence; H, hippocampus; SC, spinal Recent literature demonstrates that thyroid hormone ex- cord; BS, brain stem; BG, basal ganglia; MB, midbrain; Cbl, erts effects on the developing central nervous system cerebellum. (Adapted from Kester et al. [64]). throughout a broad period of fetal and neonatal development, and that the levels of thyroid hormone required at different stages of development are critical (5). In fact, thyroid serum iodothyronine levels. D3 activity was high in the re- hormone deficiency during development of the central ner- gions with low T3 and T4 and high rT3 levels, and low in re- vous system leads to neurologic cretinism if treatment is not gions with high T3 and T4 and low rT3 levels (Fig. 4). D3 was

started immediately after birth (59). Even undiagnosed early negatively correlated with T3 (r 0.682) and positively maternal hypothyroidism has been associated with a dimin- with rT3/T3 (r 0.812) and rT3/T4 (r 0.889) (64). In the ished neurologic outcome of the offspring (60,61). In agree- cerebral cortex, which starts to develop in the second month ment with these clinical findings, Lavado-Autric et al. (62) of gestation, D3 activity was low to undetectable. In this recently proved the sensitivity of the fetal brain to maternal brain region, T3 and D2 activity both increased during de- thyroid hormone insufficiency: even subtle insufficiency of velopment (64), whereas serum T3/T4 tended to decrease thyroid hormone in the pregnant rat disrupted the migra- during this same period (65). In contrast, in the cerebellum, tion of neurons in the fetal cortex and hippocampus, lead- where major developmental events only start at week 34 (66), ing to an altered histogenesis and cytoarchitecture of the cor- D3 activity was high and T3 was low. Cerebellar D3 started tex and hippocampus of the progeny. to decrease after midgestation (64). A coordinated regulation of brain deiodinases is critical to Because D2 catalyzes the conversion of T4 to T3, whereas maintain thyroid hormone homeostasis in the developing D3 catalyzes the conversion of T3 to 3,3 -T2 and of T4 to rT3, brain. D1 is not expressed in the human brain, D2 is ex- it can be concluded that coordinated D2 and D3 expression pressed in astrocytes and tanycytes, and D3 is expressed in deiodination is important to keep T3 concentrations in the neurons that also express nuclear T3 receptors and represent developing brain tightly regulated. D2 is responsible for the the major T3 target cells in brain (63). This suggests that as- local T3 increase in the cerebral cortex, and local D3 is im- trocytes and tanycytes take up T4 from the circulation and portant to protect brain regions from undue exposure to T3 convert it to T3. Subsequently, the T3 is transported to neu- until it is required for differentiation. rons, where the T3 exerts its actions (63). The D3 in the neurons would limit T availability according to the local tem- 3 D3 and imprinting poral needs for thyroid hormone action. In such a model a coordinated time-dependent and region-specific expression Genomic imprinting is an epigenetic process by which the of deiodinases and of thyroid hormone transporters is of cru- two parental alleles of a gene are differentially expressed ac- cial importance. cording to the parental origin (67). Two independent stud- We recently investigated the local regulation of thyroid ies, one on D3 knockout mice (68) and the other on uni- hormone in the developing human brain (13–42 weeks post- parental disomy (UPD) 12 mice (69), recently demonstrated menstrual age) by deiodinases, and showed that D2 and in that the Dio3 gene is subject to imprinting. particular D3 have key functions in this regulation (64). We D3 knockout (D3-/-) mice display remarkable growth re- found region-specific spatial and temporal patterns of D2 tardation and neonatal mortality (68), which may be because and D3 activity. Also the local iodothyronine levels showed of exposure to excessive levels of maternal thyroid hormone, spatial and temporal specificity, which indicates that these and clearly proves the key function of D3 during develop- levels could not be accounted for simply on the basis of ment. Interestingly, the heterozygous D3/ mice showed 794 KUIPER ET AL. either almost normal or strongly decreased D3 expression, Genetic Variation in Deiodinase Expression depending on wether the defective allele was inherited from the mother or the father, respectively. These data led to the Production of thyroid hormone is regulated by the classic conclusion that the D3 gene is imprinted, with preferential hypothalamic-pituitary-thyroid axis, whereas the biologic ac- expression from the paternal chromosome (68). tivity of thyroid hormone, that is, the availability of T3, is The Dio3 gene is located approximately 850-kb down- mainly regulated by the three different deiodinases (9,74). In stream of two imprinted genes, Delta-like 1 (Dlk1) and Gene- healthy subjects, serum thyroid parameters show a substantial trap locus 2 (Gtl2), on mouse chromosome 12F1 and on hu- interindividual variability, whereas the intra-individual vari- man chromosome 14q32. Tsai et al. (70) studied D3 expression ability is within a narrow range (75,76). This suggests an im- and T3 levels in UPD12 mice, that are mice that have received portant influence of genetic variation on the regulation of thy- both homologues of chromosome 12 from only one parent. roid hormone bioactivity, resulting in a thyroid function D3 mRNA expression was increased two-fold in pUPD12 em- set-point, that is different for each individual. These data are bryos and placentas compared to normal embryos and pla- supported by a classical twin study that was recently published centas, whereas in mUPD12 the level was twofold decreased (77). In this study, heritability accounted for approximately compared to normal (70). Clearly, the Dio3 gene is imprinted, 65% of the variation in serum thyrotropin (TSH), free thyrox- with preferential expression from the paternal chromosome. ine (FT4) and free triiodothyronine (FT3) concentrations. In a Disruption of the imprinting status of Dio3 in UPD12 mice Mexican American population, heritability in serum thyroid resulted in abnormal serum T3 levels: mUPD12 mice had sig- parameters ranged from 26%–64% of the total interindividual nificantly higher T3 levels than normal, and pUPD12 mice variation observed, whereas the effects of environmental co- had significantly lower T3 levels than normal, which is in variates and a broad range of lifestyle covariates accounted for agreement with the D3 expression data (70). only 2%–18% of the total phenotypic variation (78). Interestingly, patients having uniparental disomy of hu- Polymorphisms are variations in the nucleotide sequence man chromosome 14 (UPD14) have certain phenotypic char- of the genome that occur at least in 1% of a population. We acteristics, such as growth retardation (71), which are con- recently identified two polymorphisms in the D1 gene (D1a- sistent with the phenotype of UPD12 mice but also of C785T and D1b-A1814G), both located in the 3-UTR, that D3-deficient mice. Because the imprinting of Dio3 has im- are associated with serum thyroid parameters (79). The T-al- portant implications for the regulation of Dio3 expression, lele of D1a was associated in a dose-dependent manner with and disruption of Dio3 imprinting results in abnormal thy- increasing plasma rT3 levels and decreasing T3/rT3 ratio, roid hormone levels in UPD12 mice, it is likely that the im- whereas the G-allele of D1b showed an opposite tendency. printing of Dio3 contributes to the phenotypic abnormalities D1 plays a key-role in serum T3 production and in the clear- associated with UPD12 in mice and UPD14 in humans. ance of rT3, and therefore these data suggest a negative ef- Dio3 is part of a large cluster of imprinted genes, which fect of the D1a-T variant on tissue D1 activity, whereas the may all be especially important during fetal development. D1b-G variant appears to have a positive effect (79). In a dif- Other genes on the locus include Dio3as, which is a non- ferent population (350 elderly men, mean age 77 years), D1a- coding gene located only 1 kb upstream of Dio3, and is tran- T showed an allele dose effect on serum T3 concentrations, scribed antisense to the Dio3 gene (68), the paternally im- with lower T3 levels in carriers of the D1a-T variant (80). D1b- printed Dlk1 gene, which is a member of the epidermal G showed an opposite relation, which failed to reach signif- growth factor (EGF)-like protein family and plays a role in icance. Because D1 produces serum T3, this is in line with growth of several tissues (72), the supposedly noncoding our hypothesis of a decreased D1 activity in carriers of the maternally imprinted gene Gtl2, and the paternally im- D1a-T allele, but different from the correlations observed in printed retrotransposon-like gene Rtl1. The imprinting of healthy blood donors. This might be explained by the dif- Dio3 does not appear to be regulated by differential methy- ference in age between the two populations (means 46 ver- lation at CpG islands in the GC-rich Dio3 promoter (70). sus 77 years). A decreased T3 production by D1 may be However, more distant regulatory elements may contribute masked by the production of T3 by skeletal muscle D2 in to the regulation of hDio3 and other genes on the imprinted young subjects. Throughout adult life, skeletal muscle size locus. Recently, an intergenic germline-specific differen- and strength gradually decline, resulting in a decrease in D2- tially methylated region (IG-DMR) was identified on mouse expressing skeletal muscle, which is believed to contribute chromosome 12, upstream of the Gtl2 gene (73), which was to serum T3 production (9). Although D1 activity decreases found to be conserved between mouse and human (69). during aging (81,82), the relative contribution of D2 to serum Deletion of this IG-DMR from the maternally inherited chro- T3 production may be less important in elderly than in young mosome caused the bidirectional loss of imprinting of all subjects, resulting in a relatively greater contribution of D1 genes on the imprinted locus, in the case of Dio3 resulting to serum T3 production at advanced ages. in a doubling of D3 expression, because of the activation of No effect of a polymorphism in D2, D2-Thr92Ala, was ob- the normally repressed maternal allele. Because no signifi- served in blood donors (79). However, D2 plays an important cant differences in the imprinting of any of the genes were role in local T3 production in D2-containing tissues, and there- found after paternal inheritance of the deletion, this IG- fore, an effect of this polymorphism on intracellular iodothy- DMR regulates imprinting on the maternal chromosome ronine levels cannot be excluded. Mentuccia et al. (83) re- only (69). Despite these data, many essential regulatory el- ported that D2-92Ala is associated with insulin resistance, but ements for Dio3 expression remain unidentified. Further re- not with body composition in obese women. No significant search should lead to the identification of such elements that differences in D2 activity were found between the D2-92Ala regulate Dio3 imprinting or regulate its distinct expression variant and the wild-type variant, after transfection in COS pattern. cells (79). Whether this association of D2-92Ala with insulin THYROID HORMONE DEIODINATION 795

resistance may be explained by linkage with another poly- found low levels of TSH, T4, T3, and of the active over inactive morphism, and whether it can be confirmed in other popula- thyroid hormone ratio (T3/rT3), whereas we found high lev- tions, remains to be investigated in future studies. els of rT3. In livers of these patients we found D1 activity that, Until now, only one polymorphism has been identified in except for patients who died acutely from severe brain dam- the D3 gene (D3-T1546G) (79). This polymorphism was not age, was low compared to healthy individuals. Although pre- associated with changes in thyroid hormone levels in healthy liminary data suggest that D2 activity is expressed in skeletal individuals. Possible effects of this polymorphism on tissue muscle (9), no D2 activity could be detected in skeletal muscle iodothyronine levels, and possible consequences under of these patients. On the other hand, D3, which is not present pathophysiological conditions (see below), remain to be in- in liver and skeletal muscle in healthy individuals, was vestigated in future studies. A complicating aspect that may markedly induced in both liver and skeletal muscle (97). A high obscure genotype-phenotype correlations is the regulation of D3 activity decreases the T3/rT3 ratio by two ways: it prevents D3 expression by imprinting (see above). conversion of T4 to T3 by catalyzing the conversion of T4 to rT3 In conclusion, genetic variation and, thus, polymorphisms instead, and it also catalyzes the degradation of T3 to 3,3 -T2. play an important role in the interindividual variation in In these critically ill patients, liver D1 activity was positively serum thyroid function test, suggesting a unique set-point correlated with T3/rT3 ratio, whereas liver D3 tended to cor- for each individual. Subclinical hyperthyroidism and sub- relate negatively with serum T3/rT3. clinical hypothyroidism illustrate that only minor alterations It can be concluded from these data that, in addition to a in thyroid hormone bioactivity may have important conse- downregulation of D1 and possibly D2, induction of D3 quences on certain thyroid hormone-related clinical end- plays an important role during critical illness. D3 activity in points such as atherosclerosis, bone mineral density, and liver and in an abundant tissue such as skeletal muscle is heart rate (84,85). Whether polymorphic variation in deio- likely to contribute to the low serum T3 and high serum rT3 dinases will result in an altered risk for these clinical end- levels observed in critically ill patients, suggesting that the points, can now be investigated in population studies. role of D3 in thyroid hormone metabolism during criticall illness was previously underestimated. Deiodinase Expression During Critical Illness Conclusion During critical illness, pronounced alterations in plasma thyroid hormone levels occur. Plasma T3 decreases and In both qualitative and quantitative terms, deiodination is plasma rT3 increases, and the magnitude of these changes is the most important pathway of thyroid hormone metabolism. related to the severity of the disease (86). Although T4 and FT4 Since the molecular characterization of D1 in 1991, much may increase in mild illness, plasma T4 is decreased and FT4 progress has been made in the understanding of the molec- normal or decreased in critically ill patients (86). Whether the ular biology and physiology of these enzymes. One of the reduction in serum T3 is a beneficial adaptation resulting in a major achievements is the growing realization that D3 plays decreased metabolic rate, or that it is a maladaptation con- a much more important role in the regulation of thyroid hor- tributing to a worsening of the disease, is still a controversial mone bioactivity than previously assumed. This is under- issue that has been discussed extensively by others (86,87). So scored by findings of high D3 expression in fetal tissues as far, substitution of thyroid hormone in critically ill patients has well in adult tissues under pathological conditions, resulting not been shown to have a positive effect on clinical outcome, in low T3 levels not only locally but also systemically. Recent but intervention with hypothalamic-releasing factors, which findings regarding the regulation of D3 expression by im- restores pulsatile pituitary hormone secretion and normalizes printing even add more impetus to the growing status of D3. peripheral hormone levels might be a more successful ap- More surprising results of the function of D3 and the other proach (88–90). We only focus on the mechanisms behind the deiodinases are to be expected in the near future. altered iodothyronine levels. Until recently, studies on the role of deiodinases during crit- References ical illness have focused on D1 and D2. The reduction in cir- 1. Darras VM, Hume R, Visser TJ 1999 Regulation of thyroid culating T3 levels during critical illness was thought to be caused by a decreased peripheral deiodination by D1, D2, or hormone metabolism during fetal development. Mol Cell Endocrinol 151:37–47. both (9,91,92). The increase in rT3 was explained by a decreased 2. 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