Antonio C. Bianco
P. Reed Larsen
Thyroxine (T4), the main product of the thyroid gland, is a prohormone that must be activated by deiodination to triiodothyronine (T3) in order to initiate thyroid action. This deiodination reaction occurs in the phenolic or outer ring of the T4 molecule and is catalyzed by two deiodinases, type 1 (D1) and type 2 (D2) (Fig. 7.1). In humans, approximately 80% of the T3 that is produced each day is produced by extrathyroidal deiodination of T4, demonstrating the critical importance of this pathway for thyroid hormone homeostasis. In normal humans, this occurs primarily via D2, because their serum T3 concentrations do not decline much when they are given propylthiouracil (PTU), a specific inhibitor of D1. In contrast, in rodents, both D1 and D2 contribute about equally to extrathyroidal T3 production. As a counterpoint to the activation pathway, both T4 and T3 can be irreversibly inactivated by deiodination of their tyrosyl ring (inner ring deiodination), a reaction catalyzed by either D1 or type 3 deiodinase (D3), the third member of the deiodinase group. About 40% of the T4 and nearly all of the T3 that is produced each day is deiodinated by inner ring deiodination, mostly via D3 (Fig. 7.1). Therefore, the deiodinases have the capacity to promote or terminate thyroid hormone action, constituting a critical mechanism to vary thyroid status in different organs (1).
FIGURE 7.1. Structures and interrelationships between the principal iodothyronines activated or inactivated by the selenodeiodinases.
Iodine availability is likely to have been a major factor in the evolutionary pressure leading to the selection of the deiodinases as important regulators of thyroid hormone homeostasis and biologic activity. T3 is the short-lived biologically active molecule (half-life ~1 day in humans) that is responsible for most if not all thyroid action in tissues. If T3 were the main product of thyroid secretion, its production would fluctuate according to the availability of iodine. Rather, the human thyroid secretes large amounts of T4, a long-lived molecule (half-life ~7 days) that binds to circulating proteins and accumulates in a large extrathyroidal pool, approximately 770 µg (1 µmol) in adult humans. Appropriate changes in deiodinase activity accommodate variations in iodine availability by adapting to modifications in serum T4 concentrations in order to maintain constant serum T3 concentrations (1).
STRUCTURE OF THE DEIODINASES
The three deiodinase proteins (D1, D2, and D3) are structurally similar (~50% sequence identity). All are integral membrane proteins of 29 to 33 kd, and they are especially similar in the region surrounding the active catalytic center (2,3,4). Because of inherent difficulties in the crystallization of membrane-anchored proteins, further insight into the structures of these proteins has been obtained through the use of hydrophobic cluster analysis (5), which is based on protein folding and two-dimensional transposition of sequences, allowing resolution of a sequence into its secondary structures centered on the so-defined hydrophobic clusters.
Based on this type of analysis, the three deiodinases have a single transmembrane segment, which is present near the N-terminus, and several well-conserved clusters, typical of α-helixes or β-strands, suggesting that they correspond to core secondary structures of the deiodinases. A striking common feature is the similarity of the deiodinases with various members of the thioredoxin (TRX) family, proteins that contain in their three-dimensional structure a fold defined by βαβ and ββα motifs (Fig. 7.2). The deiodinases contain extra elements, not found in the canonical thioredoxin fold, that interrupt the relationship between the βαβ and ββα motifs. These intervening sequences correspond to distinct secondary structural elements added to the canonical thioredoxin fold, a feature also present in other proteins of the thioredoxin family (6). A unique aspect of the deiodinases, however, is that one of these intervening sequences has striking similarity to α-L-iduronidase (IDUA; 47% identity with D1 and D3 and 60% with D2), a lysosomal enzyme that cleaves α-iduronic acid residues from the glycosaminoglycans heparan sulfate and dermatan sulfate (7) (Fig. 7.2). This structural similarity between the deiodinases and iduronidase may reflect the similarity of their substrates, T4 (or T3) and sulfated α-L-iduronic acid, respectively.
FIGURE 7.2. Three-dimensional structures and organization of secondary structure of the arche type thioredoxin enzyme and the rough model of the deiodinases deduced from the alignment revealed by hydrophobic cluster analysis. The transmembrane, hinge, and intervening sequences are circled. (Modified from Callebaut I, Curcio-Morelli C, et al. The iodothyronine selenodeiodinases are thioredoxin-fold family proteins containing a glycoside hydrolase-clan GH-A-like structure. J Biol Chem 2003;278:36887, with permission.)
The three-dimensional model predicts that the active center of the three deiodinases is a pocket defined by the β1-α1-β2 motifs of the thioredoxin fold and the IDUA-like insertion (Fig. 7.3). The most striking feature of this pocket is the presence of the rare amino acid selenocysteine, which is critical for the catalytic activity of the three deiodinases. The presence of selenocysteine was first identified when rodent D1 complementary DNA (cDNA) was found to contain UGA that encoded selenocysteine; in the vast majority of messenger RNAs (mRNAs), UGA is a stop codon (8). The cis-acting sequences that allow for the incorporation of selenocysteine rather than a stop codon consist of the seleocysteine codon (UGA) itself and a specific RNA stem-loop located in the 3′-untranslated region of the mRNA. Only in the presence of the stem-loop structure and in several trans-acting factors are UGA codons “recoded” to specify selenocysteine. The stem-loop sequence is termed the Selenocysteine (sec) insertion sequence, or SECIS, element; it is present in all three deiodinases and all other eukaryotic selenoproteins (9)
FIGURE 7.3. Schematic representation of the putative active site of deiodinases deduced from sequence alignment and from the associated modeling (see Fig. 7.2). The positions shown are those of type 2 deiodinase (D2), and the table contains the corresponding positions and residues in D1 and D3. The iduronidase (IDUA)-like insertion likely constitutes a cap that may cover the active site after ligand binding. (Modified from Callebaut I, Curcio-Morelli C, Mornon JP, et al. The iodothyronine selenodeiodinases are thioredoxin-fold family proteins containing a glycoside hydrolase-clan GH-A-like structure. J Biol Chem 2003;278:36887, with permission.)
All three deiodinases can form homodimers when transiently expressed (10,11) Fig. 7.4. This is supported by the finding of catalytic activity in the higher-molecular-weight forms (~65 kd) (12,13). However, these higher-molecular-weight forms of the deiodinases may reflect associations with other cellular proteins not primarily involved in their catalytic function, but which could, for example, regulate their half-life, intracellular transport, or subcellular localization. It is not clear that dimerization occurs or that it is necessary for function when the enzymes are expressed at endogenous levels.
FIGURE 7.4. Subcellular localization and topology of D1, D2, and D3. Left panel: Immunofluorescence confocal microscopy of HEK-293 cells transiently expressing one of the deiodinases as indicated. D1 is visible in the periphery of the cell, and does not colocalize with BiP, an endoplasmic reticulum marker visible around the nucleus. D2 is visible colocalized with BiP, whereas D3 is visible in the cell border. Right panel: Schematic representation of type 1 deiodinase (D1), D2, and D3 dimers with their respective orientations and subcellular localization. (From Toyoda N, Berry MJ, Harney JW, et al. Topological analysis of the integral membrane protein, type 1 iodothyronine deiodinase (D1). J Biol Chem 1995;270:12310; Baqui MM, Gereben B, Harney JW, et al. Distinct subcellular localization of transiently expressed types 1 and 2 iodothyronine deiodinases as determined by immunofluorescence confocal microscopy. Endocrinology 2000; 141:4309; and Baqui MM, Botero D, Gereben B, et al. Human type 3 iodothyronine selenodeiodinase is located in the plasma membrane and undergoes rapid internalization to endosomes. J Biol Chem 2003;278:1206, with per mis sion.)
TOPOLOGY AND SUBCELLULAR LOCALIZATION
The D1 and D2 monomers are integral membrane proteins oriented with a small amino-terminal extension in the extracellular space (D1) or lumen of the endoplasmic reticulum (D2), and a single transmembrane domain exiting the membrane at about position 40 (14,15). This puts the active center of both D1 and D2 in the cytosol. D3 is also an integral membrane protein, but its orientation is opposite, so that its active center is the extracellular space Fig. 7.4.
The location of mature D1 in the plasma membrane has been demonstrated in several types of cells that normally contain D1, including thyroid and kidney cells, and in cells transiently expressing the enzyme (10,16,17,18). Using confocal laser microscopy of human and mouse cells, transiently expressed FLAG-tagged D1 was also localized to the plasma membrane. D1 does not colocalize with the endoplasmic reticulum protein BiP, as does D2 (15). This plasma membrane localization has been confirmed by labeling of D1 with cell-impermeable reagents (19). D2, on the other hand, is an endoplasmic reticulum protein. Immunofluorescent confocal microscopy of human and mouse cells transiently expressing FLAG-tagged D2 revealed colocalization of D2 with BiP. Endogenously expressed D2 also colocalizes with BiP in MSTO-211H cells (20).
Using similar techniques, endogenously and transiently expressed FLAG-tagged D3 was identified in the plasma membrane. It colocalizes with the α-subunit of Na+, K+-ATPase, the early endosomal marker EEA-1, and clathrin, but not with endoplasmic-reticulum proteins. Most of the D3 is extracellular (Fig. 7.4), and its extracellular portion can be covalently labeled with a cell-impermeant probe, confirming its plasma membrane location. There is constant internalization of D3 that is blocked by sucrose-containing medium, and exposing cells to a weak base such as primaquine increases the pool of internalized D3, suggesting that D3 is recycled between plasma membrane and early endosomes. Such recycling could account for the longer half-life of D3 (12 hours), as compared with D1 (8 hours) or D2 (1 hour) (19).
A plasma membrane location for D1 could be viewed teleologically as allowing ready access of circulating T4 to the enzyme as well as facilitating the entry of the T3 produced from T4 into the extracellular fluid. The plasma membrane localization of D1 contrasts with the localization of D2 in the endoplasmic reticulum in the same cell types (15). This differential subcellular localization of D1 and D2 may explain the small contribution of T3 generated by D1 and the large contribution of T3 generated by D2 to intranuclear T3 (21,22,23,24). The extracellular location of D3 makes it readily accessible to extracellular, and therefore circulating T4 and T3, explaining its capacity for rapid inactivation of circulating T4 and T3 in patients with hemangiomas and its blockade of the access of maternal thyroid hormones to the fetus.
STRUCTURE-FUNCTION RELATIONSHIPS OF DEIODINASES AND MECHANISM OF DEIODINATION
The deiodination of iodothyronines by D1, D2, and D3 is a reductive dehalogenation that in vitro requires a reducing agent, such as dithiothreitol, as cofactor. An endogenous cofactor capable of sustaining multiple rounds of iodothyronine deiodination has not yet been identified. Based on major differences in substrate preference, reaction kinetics, and sensitivity to various inhibitors, it was assumed that the substrate-binding pockets of these enzymes were fundamentally different. However, the structural model described above predicts a conserved structure for all three deiodinases, particularly for the active center, the accuracy of which is supported by the substantial perturbations in enzyme function that result from changes of single amino acids in the binding pocket (5) (Fig. 7.3).
D1 has a relatively low affinity for T4[Michaelis-Menten constant (Km), 1,2 µM], and its catalytic activity is bisubstrate in nature, with a thiol-containing cofactor serving as the second substrate with “ping-pong” kinetics (25) Fig. 7.5.D2 and D3, on the other hand, have relatively high affinity for T4 and T3, respectively (Km, 1–4 nM), and both have sequential reaction kinetics, suggesting that the iodothyronine and the thiol-containing cofactor interact with the enzyme simultaneously before the reaction takes place (25). Regardless of the reaction kinetics, the selenocysteine residue in the active center of the three deiodinases probably acts as a nucleophile catalyzing the removal of iodine. The critical role of selenocysteine in this function was ascertained through characterization of the kinetic properties of the wild-type selenoenzymes (D1, D2, and D3) and the corresponding cysteine (Cys) mutants, which have lower affinity for the substrates and decreased turnover rates (2,11,26).
FIGURE 7.5. Mechanism of type 1 deiodinase (D1)-catalyzed thyroxine (T4) conversion to triiodothyronine (T3). The steps in the enzymatic reaction cycle at which iodoacetic acid (IAc) and propylthiouracil (PTU) are thought to inhibit catalysis are indicated. (From Leonard JL, Rosenberg IN. Thyroxine 5′-deiodinase activity of rat kidney: observations on activation by thiols and inhibition by propylthiouracil. Endocrinology 1978;103:2137, with permission.)
D1, D2, and D3 differ in their sensitivity to PTU. D1 is quite sensitive [inhibition constant (Ki), 5 µM; in vitro at 10 µM dithiothreitol], but D2 and D3 are not (Ki, >1 mM). PTU probably inhibits D1 by competing with the endogenous thiol-containing cofactor for a putative selenenyl iodide (E-Se-I) intermediate (25) Fig. 7.5. Supporting this interpretation is the fact that PTU inhibition is uncompetitive with the first D1 substrate (iodothyronine), but competitive with the second (e.g., dithiothreitol) (25). Because of the PTU insensitivity, D2- or D3-catalyzed deiodination proceeds by removal of an iodonium (I+) ion by the endogenous cofactor, resulting in an enzyme-thyronine intermediate [D2-T3 (or reverse T3)] complex and a cofactor-Se-I complex (27). In this regard, it is notable that the sequence Thr-Sec-Pro-Pro/Ser-Phe is identical in D1, D2, and D3, but that in all D1 sequences, excepting the PTU-insensitive D1 of the blue tilapia (Oreochromis aureus), the uncharged polar side chain of the Ser residue substitutes for the nonpolar side chain of Pro at position 128 (28). The existence of this natural variation prompted the creation of the Ser128Pro D1, Pro135Ser D2, and Pro146Ser D3 proteins Fig. 7.3. Remarkably, replacement of Pro135 with Ser in D2 results in a two orders of magnitude increase in Km (T4) to approximately 250 nM, approximately 10-fold lower than that of D1 for T4, and the enzyme operates with ping-pong kinetics. Furthermore, the Pro135Ser D2-catalyzed deiodination is two orders of magnitude more sensitive to PTU (Ki, 4.0 µM), although PTU inhibition is noncompetitive with dithiothreitol. Thus, the substitution of Ser for Pro135 in D2 results in changes in the enzyme that make its kinetics more similar to those of D1, indicating a critical influence of the amino acid occupying this position on enzyme function. Similar observations were made with the Pro146Ser D3 protein, which has a fivefold higher Km (T3) and is highly sensitive to inhibition by PTU (Ki, 1.0 µM). As mentioned above, Ser128Pro D1 catalyzed deiodination is resistant to PTU (Ki, >1 mM), suggesting that there is no longer an accessible E-Se-I intermediate, again illustrating the pivotal role of this position in the active center of the deiodinase molecule.
The presence of Pro in the 128/135/146 positions of D1, D2, and D3 may result in tighter binding of the substrate in the D2 (and D3) binding pockets, perhaps explaining the approximately 1000-fold higher affinity of D2 and D3 for T4, as compared with D1. This could reflect an interaction between the phenolic hydroxyl group of T4 and the Pro residue at these positions, and explain why sulfate conjugation of the phenolic hydroxyl group dramatically increases the maximum velocity (Vmax)/Km and changes the T4 deiodination site from an outer to an inner ring iodine (29).
Three conserved amino acids with charged polar side chains mark the transition between the β2-strand and the α-helix in the IDUA-like insertion, Glu/Asp155, Glu156, and His158 (D1 residues). When the invariant Glu156 (D1), Glu163 (D2), and Glu174 (D3) were replaced with Ala, the resulting enzymes had no deiodinase activity. Replacement of Glu156 with Asp in D1 supported deiodination, but with an approximately 4.5-fold higher Km (rT3), whereas a similar substitutio at position 163 in D2 did not alter the Km (T4). Thus, the acidic amino acids in this region of the deiodinase pocket are important for substrate binding or enzyme function, although the length of the side chain can vary. This is further supported by mutational studies of His at position 158 in D1 (30). Its mutation to Asn, Gln, or Phe resulted in complete loss of deiodinase activity. Replacement of the corresponding His165 in D2 with Asn also resulted in loss of deiodinase activity. According to the model, residues in this acidic pocket could interact with either the amino or the carboxyl group in the alanine side chain of the iodothyronines, a hypothesis supported by previous studies indicating that the positively charged T4 analogue (3,5,3′,5′-tetraiodothyroethylamine), which lacks a carboxyl group, is not a substrate for D1 (31). In addition, the compounds with the highest affinity for D1 (lowest apparent Km values) are those that lack positively charged functional groups (NH3+), such as tetra iodothy roacetic acid. Furthermore, the Km (T4) values for D- and L-T4 are similar (31). These results argue that the carboxyl group in the iodothyronines interacts with the NH3 group of His in the 158 position of D1 and that the other acidic residues in this pocket act to reduce the ionization of the His residue. The critical role played by the IDUA-like insertion is further strengthened by the complete loss of deiodinase activity when the conserved Trp163 in D1 and the corresponding Trp170 in D2 are replaced with Ala.
SPECIFIC PROPERTIES OF THE DEIODINASES
Type 1 Deiodinase
PTU-inhibitable, D1-catalyzed conversion of T4 to T3 supplies approximately 20% to 30% of the T3 in the serum in normal humans, but 50% or more in patients with thyrotoxicosis. D1 is the only selenodeiodinase that can function as either an outer (5′)- or inner (5)-ring iodothyronine deiodinase, D2 and D3 being exclusively outer and inner ring deiodinases, respectively (32). The molecular basis for these differences is not known.
The gene (Dio1) for human D1 is on chromosome 1 p32-p33, in a region syntenic with mouse chromosome 4, the location of mouse Dio1 (33). The complete cDNA sequences have been determined for rat, human, mouse, dog, chicken, and tilapia D1 (8,28,34,35,36,37). The size of the mRNAs for these D1s is about 2 to 2.1 kb, and all contain a UGA codon in the region encoding the active center, which is highly conserved among species. By Northern analysis, D1 is expressed in many tissues of most vertebrates, but not in amphibia (38,39,40). In rats, these include the liver, kidneys, central nervous system, anterior pituitary gland, thyroid gland, intestine, and placenta. In humans, D1 activity is notably absent from the central nervous system, but is present in the liver, kidneys, thyroid and pituitary (41,42).
Regulation of D1 Synthesis
Changes in D1 activity have been investigated in developing rats; in general, D1 activity is low in all tissues of fetal rats. It appears soon after birth in the intestine, liver, kidneys, cerebrum, cerebellum, and gonads (43). The age- related changes in D1 mRNA content are similar, indicating that the changes in activity arise at a pretranslational level. The mechanism for the age-related changes in D1 expression is unknown. The physiologic benefit of the low D1 activity in the fetus is presumably low serum T3 concentrations, thus permitting changes in intracellular T3 to be determined by the developmentally programmed changes in D2 and D3 activity (44).
Thyroid hormone–induced increases in D1 activity and mRNA content are well documented in rats, mice, and humans (8,45). The increases are due to increased transcription, which in the human Dio1 gene can be attributed to the presence of two thyroid hormone-response elements (TREs) in the 5′-flanking region of the gene (46,47,48). Studies in thyroid receptor (TR) knockout mice indicate that the T3-induced D1 stimulation is largely mediated by the β subtype of the receptor (49). Given this finding, the responsiveness of the human Dio1 gene to T3 would be expected to be greatest in patients with thyrotoxicosis. In fact, the D1 mRNA content of peripheral blood mononuclear cells is increased in proportion to the degree of thyrotoxicosis (42). This increase can explain the acute decrease in serum T3 concentrations that occurs in response to PTU in patients with thyrotoxicosis (50).
Interleukin-1 (IL-1), IL-6, tumor necrosis factor-α (TNF-α), and other cytokines are potential mediators of the alterations in thyroid function that occur in patients with severe nonthyroidal illness (see section on nonthyroidal illness in Chapter 11) (51,52,53). TNF-α, IL-1β, and interferon-γ decrease D1 activity and mRNA in rodent thyroid (FRTL5) cells (54). The effects of TNF-α have been examined in hepatocytes and HepG2 cells with contradictory results. TNF-α decreased the stimulatory effect of T3 on D1 mRNA production in HEPG2 cells, an action that is blocked by nuclear factor kappa B (55). In dispersed rat hepatocytes, IL-1βand IL-6 blocked T3 stimulation of D1 mRNA and activity; however, TNF-α had no effect (56). The effect of IL-1βwas blocked by coexpression of the nuclear steroid receptor coactivator-1 (SRC-1), but not by CREB binding protein (CBP) or CBP-associated factor (pCAF). Because IL-1 did not alter the amount of SRC-1 in hepatocytes, the effect was attributed to competition between IL-1 and T3-stimulated transcriptional events for limiting quantities of SRC-1. More recent studies describing decreases in D1 activity in liver and D2 activity in skeletal muscle and increases in D3 activity in liver and skeletal muscle in patients dying of multiple organ failure and related conditions are discussed in the section on nonthyroidal illness in Chapter 11.
Nutritional Influences on D1 Expression
A decrease in serum T3 concentrations relative to those of T4 and an increase in serum reverse T3 concentrations during fasting in humans was one of the earliest indications that the peripheral metabolism of thyroid hormones in humans was modulated by physiologic or pathophysiologic events (57). Similar changes occur in virtually all acutely ill and many chronically ill patients (58,59). Because thyroidal secretion accounts for only about 20% of daily T3production in humans, the illness-associated decrease in serum T3 concentrations must be caused, largely if not completely, by decreased T4 to T3 conversion by D1 or D2 or by increased T3 clearance by D3 (60,61).
Early studies of liver D1 activity in rats suggested that the decrease in T4 conversion to T3 that occurred during fasting might be caused by a decrease in the thiol cofactor that serves as the cosubstrate for D1-catalyzed T4 to T3 conversion (62,63). However, this cofactor has not been identified. Although rats have been studied extensively as a model for the effects of fasting (and illness) on T4 to T3 conversion in humans, they are a poor model for the effects of fasting in humans because of their low body fat content and the fact that, unlike humans, their serum TSH and T4 concentrations decrease rapidly when they are starved (64). Also, despite reduced hepatic D1 activity, total body conversion of T4 to T3 is not reduced during starvation in rats (65,66). The marked fasting- induced reduction in serum TSH, T4, and T3 concentrations (i.e., central hypothyroidism) in rats is probably due, at least in part, to leptin deficiency (67). Prefeeding rats with a high-fat diet to induce obesity results in less urinary nitrogen loss and a lesser decline in serum T4 and T3 concentrations during starvation, and serum T3 concentrations actually increase if the period of starvation is prolonged (64). In contrast, in humans, serum T3 concentrations decrease rapidly to about 50% of baseline during fasting, and they remain low for up to 3 weeks of fasting, but serum T4 and TSH concentrations change little (68).
A decrease in hepatic D1 activity of Se-deficient rats and the demonstration that D1 could be labeled with 75Se were the first clues that this trace element was critical to the function of D1 (69,70,71,72). However, the effects of Se deficiency on the synthesis of intracellular selenoproteins, such as the selenodeiodinases, depend on the tissue being examined. For example, in Se-deficient rats, thyroidal D1 activity is preserved, while that in the liver declines precipitously (73), serum T4 concentrations increase, and serum T3 concentrations do not change (74). Se deficiency also decreases D1 activity in the kidneys; this is accompanied by a decrease in D1 mRNA, which does not occur in the liver (75). Se deficiency can occur in patients receiving diets that are restricted in protein content, such as those given for phenylketonuria, and has also been found in elderly patients (76,77,78,79). In Se-deficient humans, serum T4concentrations and the serum ratio of T4 to T3 are slightly increased, but serum TSH concentrations are normal. In one endemic goiter region in Africa, there is an accompanying Se deficiency (80,81). When Se was supplied to these iodine-deficient people, their thyroid function deteriorated, as evidenced by an increase in serum TSH concentrations and a decrease in serum T3 concentrations, suggesting that the reduction in D1 activity during Se deficiency can protect against iodine deficiency, presumably by reducing inner ring deiodination of T4 or T3 (82,83).
Type 2 Deiodinase
Type 2 deiodinase is an obligate outer ring selenodeiodinase that catalyzes the conversion of T4 to T3. The Km of D2 for T4 is in the nanomolar range under in vitro conditions in the presence of 20 mM dithiothreitol. The presence of considerable D2 activity in human skeletal muscle, unexpected because it is absent in rats and mice, provides a plausible source for a substantial fraction of the extrathyroidally generated T3 in human serum (26). T4 causes a posttranslational decrease in D2 activity, due to stimulation of proteolysis of D2 via the ubiquitin-proteasome pathway (84,85,86).
Gene Structure and Chromosomal Localization
The Dio2 gene is present as a single copy located on the long arm of chromosome 14 (14q24.3) in humans (87,88). It is about 15 kb in size, and the coding region is divided into two exons by an intron of approximately 7.4 kb. The exon/intron junction is located in codon 75, and is at the same position in the human and mouse Dio2 genes (87,89,90). For the human gene, there are three transcriptional start sites, 707, 31, and 24 nucleotides 5′ to the initiator codon ATG. The longest 5′-untranslated region of human D2 mRNA contains an intron of approximately 300 bp that can be alternatively spliced (90). Other splicing variants involving the coding region have also been identified (91). The human, mouse, and rat Dio2 5′-flanking regions have been isolated and functionally characterized. All contain a functional cyclic adenosine monophosphate (cyclic AMP) response element, but only human Dio2 has binding sites for thyroid transcription factor-1 (TTF-1) (90,92,93).
D2 mRNA and Protein
Human, mouse, and chicken D2 cDNAs containing intact 3′-untranslated regions (5- to 7.5-kb) have been identified using GenBank searches and library screening. These D2 cDNAs encode functional D2 proteins, as determined by expression in Xenopus laevis oocytes (89,94,95). Rat and human D2 mRNAs are approximately 7.5 kb, and chicken cDNA is approximately 6 kb (93,94,95,96). A detailed analysis involving nuclease mapping, primer extension, and Northern blots indicated that human D2 mRNA exists as four different transcripts in thyroid, brain, and possibly other tissues (90). The longest transcript is approximately 7.5 kb, starts 708 nucleotides up-stream from the initiator ATG codon, and is the only transcript found in placenta. A shorter (approximately 7.2-kb)-minor D2 species uses the same transcriptional start site, but the approximately 300-bp intron is spliced out. Two shorter transcripts of approximately 6.8 kb, differing by only seven nucleotides, use 3′ transcriptional start sites close to the translation initiation site. It is not known whether the rat and mouse genes use the same two ma jor transcriptional start sites, but this is likely to be so for D2 in rat brain (26,90,96). The deduced amino acid sequences of the chicken, mouse, rat, and human D2 enzymes contain two selenocysteine residues. The first is in the active center of the enzyme, whereas the second is located close to the carboxy-terminus. In fish and frog D2, there is only one selenocysteine codon, which is located in the active center of the enzyme (26,89,95,96,97,98). Truncating the C-terminal amino acids, including the C-terminal selenocysteine, in human D2 has no effect on D2 enzyme kinetics or activity (99).
In rats, D2 activity is predominantly expressed in the pituitary, brain, and brown adipose tissue (24,100,101,102,103,104. D2 activity is also present in the gonads, pineal, thymus, and uterus of rats, mammary gland of mice, and vascular smooth muscle cells in humans (43,105,106,107,108,109). High levels of D2 mRNA and activity are found in the mouse cochlea at the eighth postnatal day, suggesting a role for D2 in generating T3 for cochlear development (110). In the cerebral cortex of neonatal rats, D2 mRNA is present in astrocytes (111) and tanycytes; the latter are specialized ependymal cells lining the third ventricle that have multiple cellular processes that express D2 mRNA and that extend to the median eminence (111,112,113,114). A monosynaptic pathway has also been identified between the arcuate nucleus, which contains D2, and the paraventricular nucleus, which contains thyrotropin-releasing hormone (TRH) (115).
In humans, D2 mRNA or activity is expressed not only in vascular smooth muscle cells, but also in the thyroid, heart, brain, spinal cord, skeletal muscle, and placenta, and small amounts of D2 mRNA have been detected in the kidneys and pancreas (26,90,96,116,117,118). Thyroid tissue contains relatively more D2 mRNA than D2 activity, with the exception of thyroid tissue in patients with thyrotoxicosis caused by Graves' disease and follicular adenomas, in which both D2 mRNA and activity are present in large amounts (116). The discrepancy between D2 mRNA and activity is probably due to substrate-induced D2 ubiquitination. D2 mRNA sequences are also present in libraries from prostate, breast, and uterus, but none of these tissues have D2 activity (94). D2 mRNA or activity is present in human pituitary glands and brain tumors (116,119,120), and D2 activity has been found in human keratinocytes (121) and mesothelioma cells (20).
Regulation of D2 Synthesis
The Dio2 gene is regulated in part by a cyclic AMP-mediated pathway. Cold exposure increases D2 mRNA and activity in brown adipose tissue in rodents, and α1- or β-adrenergic antagonist agents block this effect (104,122). In isolated brown adipocytes, the increase of D2 activity during catecholamine treatment is actinomycin D sensitive (123,124,125). In addition, D2 activity in brown adipose tissue is induced by norepinephrine, isoproterenol, insulin, and glucagon, and it is inhibited by growth hormone (126,127). Cyclic AMP increases D2 activity in mesothelioma cells (20) and in rat astroglial cells (101,128), as does both nicotine and cyclic guanosine monophosphate (cyclic GMP) (129,130). As noted above, D2 mRNA and activity are increased in thyroid tissue from patients with Graves' thyrotoxicosis, and forskolin increases D2 mRNA in dispersed human thyroid cells (90,116). It is therefore not surprising that human, rat, and mouse Dio2 contains a cyclic AMP response element approximately 90 nucleotides upstream of the transcriptional start site (90,92,93,131). The promoter activity of human Dio2 increases 10-fold when cells are cotransfected with Dio2 and the α-catalytic subunit of protein kinase A. Mutation of the latter element abolishes the effect and decreases basal expression of Dio2 by approximately 90% (90).
Although there is a high level of D2 mRNA in human thyroid tissue, no D2 mRNA or activity is present in FRTL-5 rat thyroid cells, and in adult rat thyroid tissue D2 mRNA levels are very low and D2 activity is undetectable (93,116,132). Expression of the Dio2 gene in human thyroid tissue is under the control of TTF-1 but is not affected by Pax-8 (93). The human Dio2 gene has two TTF-1 binding sites, which are not present in the rat Dio2 gene, despite an overall 73% cross-species homology. The lack of these sites may explain the very low expression of D2 mRNA and activity in rat thyroid tissue.
Regulation of Degradation of D2
D2 is the critical T3-generating deiodinase due to its substantial responsiveness to physiologic signals. For example, D2 responsiveness to cyclic AMP constitutes the basis for the adrenergic stimulation of D2 activity in brown adipose tissue, and human skeletal muscle and thyroid tissue. This links D2 expression with the sympathetic nervous system and widens the spectrum of environmental and endogenous stimuli that can potentially influence adaptive T3production (see reference 1 for review).
Several transcriptional and posttranslational mechanisms have evolved to ensure tight control of tissue levels of D2, which is inherent to its homeostatic function. The D2 mRNA in higher vertebrates is more than 6 kb in length, containing long 5′ and 3′ untranslated regions. The D2 5′ untranslated regions are greater than 600 nucleotides in length, and they contain three to five short open reading frames, which reduce D2 expression by as much as fivefold (133). Alternative splicing is another mechanism that regulates the level of D2 synthesis, because mRNA transcripts similar in size to the major 6- to 7-kb D2 mRNAs, but not encoding an active enzyme, are present in both human and chicken tissues (133).
The ratios of D2 activity to D2 mRNA level in tissues vary, indicating substantial posttranslational regulation of D2 expression (134). In fact, the decisive property of D2 that characterizes its homeostatic behavior is a half-life of approximately 40 minutes that can be further reduced to approximately 25 minutes by exposure to physiologic concentrations of its substrate, T4, or extended to approximately 300 minutes when cells are grown in medium lacking T4(135,136,137,138,139,140,141,142). This constitutes a rapid, potent regulatory feedback loop that efficiently controls T3 production and intracellular T3concentrations based on how much T4 is available. The potency of the T4 in inducing loss of D2 activity mirrors the enzyme's affinity for the substrate, indicating that enzyme–substrate interaction must occur in order to induce loss of D2 activity.
At the molecular level, D2 activity is regulated by conjugation to ubiquitin, a protein of approximately 8 kd. The ubiquitinated D2 is subsequently recognized and degraded by proteasomes (143,144) (Fig. 7.6). The first evidence for this process was obtained in GH4C1 cells, in which the half-life of endogenous D2 was noted to be stabilized by MG132, a proteasome inhibitor (84). Substrate-induced loss of D2 activity was also inhibited by MG132 in these cells, indicating that both pathways affecting loss of D2 activity were mediated by the proteasomes. This implies that the loss of D2 activity is at least partially due to proteolysis of D2, a premise that was confirmed when the levels of immunoprecipitable D2 were found to parallel D2 activity, both under basal conditions and after exposure to T4 (85). In subsequent studies it became clear that D2 is ubiquitinated (86), and the various enzymes involved in this process were identified. In studies in which human D2 was expressed in yeast, Ubc6p and Ubc7p were identified as the ubiquitin conjugases involved in ubiquitination of D2 (145), and it is now clear that these conjugases play a role in ubiquitination of human D2 (146,147).
FIGURE 7.6. Representation of ubiquitination and proteasomal degradation of type 2 deiodinase (D2). Ub, ubiquitin; E1, enzyme that activates Ub; E2, Ub conjugase; Ubc6 and Ubc7, E2s involved in D2 ubiquitination; Cue1, endoplasmic reticulum–docking protein for Ubc7; asterisk in the D2 molecule, selenocysteine-containing active center; isopeptidase catalyzes D2 deubiquitination.
Fusion of the 8–amino acid FLAG sequence to the carboxyl-, but not the amino-, terminus of D2 prolongs its activity and increases the size of the ubiquitin-D2 pool by 20- to 30-fold (86), suggesting that D2 ubiquitination is reversible, because not all Ub-D2 undergoes proteolysis. Enzymatic deubiquitination of ubiquitin-D2 occurs in vitro (148) and could explain recycling in vivo. D2 was recently identified as a substrate for the deubiquitinating enzymes VDU1 and VDU2 (149). Confocal studies indicate that both VDUs colocalize with D2, itself an integral endoplasmic-reticulum membrane protein. The physical colocalization of VDU with D2 provides the opportunity for deubiquitination of D2.
VDU1-catalyzed D2 deubiquitination is an important part of the adaptive mechanism that regulates thyroid hormone action. In stimulated brown fat tissue, D2 increases intracellular T3 production, resulting in isolated tissue thyrotoxicosis (150,151,152). This is an important mechanism for cold acclimatization in rodents; mice with targeted inactivation of the D2 gene develop hypothermia and marked weight loss during cold exposure due to impaired thermogenesis in brown adipose tissue (151,153). Increased VDU1-catalyzed deubiquitination of ubiquitin-D2, and therefore rescue of D2 from proteasomal degradation, is an integral part of this mechanism. In brown adipose tissue, VDU1 mRNA levels are markedly up-regulated by cold exposure or norepinephrine, which amplifies the transcriptional increase in D2 activity, and hence T3 production increases by approximately 2.5-fold. Although ubiquitination is known to play a physiologic role in several cellular processes (154,155,156,157,158), enzyme reactivation due to deubiquitination is unusual.
The availability of a reversible ubiquitination-dependent mechanism to control the activity of D2 constitutes a biochemical and physiologic advantage that allows for rapid control of thyroid hormone activation. The finding that VDU1 and VDU2 are coexpressed with D2 in many human tissues, including brain, heart, and skeletal muscle (1,159), suggests that the importance of this mechanism may extend well beyond thermal homeostasis to include brain development, cardiac performance, glucose utilization, and energy expenditure.
Type 3 Deiodinase
D3, acting by inner ring deiodination, is the major T3- and T4-inactivating enzyme, although D1 also has some activity as an inner ring deiodinase (160). D3, which has almost exclusively inner ring deiodination activity, catalyzes the conversion of T4 to reverse T3 and the conversion of T3 to 3,3′-diiodothyronine (T2), both of which are biologically inactive Fig. 7.1. That reverse T3 and T2 do not support thyroid hormone–dependent gene expression is illustrated by the severe consumptive hypothyroidism that occurs in patients with hemangiomas, in whom tumor overexpression of D3 results in very high serum reverse T3 concentrations, and the blockade of metamorphosis that occurs in tadpoles overexpressing D3 (161,162).
D3 contributes to thyroid hormone homeostasis by protecting tissues from an excess of thyroid hormone. It was identified in monkey hepatocarcinoma cells (NCLP6E), and the first physiologic studies were performed in the central nervous systems of rats (163,164,165,166). In humans, D3 is present in not only the central nervous system, but also skin and placenta; it is also present in fetal liver and in the uterus of pregnant rats (167). The highest activity found to date is in hemangioma-type tumors in humans (162). In amphibians, D3 plays a critical role in development (168); it is present in tadpoles from premetamorphosis to the onset of the metamorphic climax, after which it declines to barely detectable levels. In mammals, D3 is critical for thyroid hormone homeostasis, because it protects the fetus from premature exposure to excessive amounts of thyroid hormone, which can result in malformations, altered growth, mental retardation, and even death. In fetal and neonatal animals, D3 expression is highly regulated in tissue-specific patterns that are likely to be critical to the coordinated regulation of thyroid hormone effects on development.
Gene Structure and Chromosomal Localization
The Dio3 gene is located on human chromosome 14q32 and mouse chromosome 12F1 (169). A unique feature of the human and mouse Dio3 gene is that it has no introns (3), which is a rarity among eukaryotes (169,170). The gene is preferentially expressed (imprinted) from the paternal allele in mice (171). The Dio3gene likely belongs to the same cluster of imprinted genes in mouse chromosome 12 and human chromosome 14, and as such it might play a role in the phenotypic abnormalities associated with uniparental disomy of those chromosomes, a condition in which gene expression is altered due to abnormal genomic imprinting (172).
Human D3 mRNA contains 2066 nucleotides. There are 220 bp in the 5′-untranslated region, an 834-bp open reading frame, and a 3′-untranslated region of 1012 bp (173). All D3 cDNAs identified to date include a selenocysteine-encoding TGA codon, as well as SECIS element in the 3′-untranslated region. There is a high degree of identity between the Dio3 gene in human and other species, particularly in the putative active center where the selenocysteine is located. The conservation of this enzyme from tadpoles to humans implies that its role in regulating thyroid hormone inactivation during embryologic development is essential. The most common form of D3 mRNA in most tissues is 2.3 kb, but there are at least four differently sized mRNAs in the central nervous system of rats, and thyroid hormone causes increases in the relative intensity of these mRNAs (174).
D3 has been identified in various tissues in several animal species, among which rats have been studied most extensively. In adult rats, D3 is found predominantly in the central nervous system and skin. In neonatal rats, it is found not only in these tissues, but also in skeletal muscle, liver, and intestine (43,164,175,176,177,178). In particular, using in situ hybridization analysis, D3 mRNA has been identified throughout the brain in adult rats, especially in hippocampal pyramidal neurons, granule cells of the dentate nucleus, and layers II to VI of the cerebral cortex (174). It is noteworthy that these regions also contain the highest con centrations of thyroid receptors in the brain, and they have critical roles in learning, memory, and higher cognitive functions (179,180,181). Furthermore, the distribution of D3 mRNA in the central nervous system changes during the early stages of development. At postnatal day 0, D3 is selectively expressed in the bed nucleus of the stria terminalis, the preoptic area, and other areas related anatomically and functionally to the bed nucleus, such as the central amygdala; all of these areas are involved in the sexual differentiation of the brain (182). D3 expression in these areas was transient and was no longer detected at postnatal day 10. The overall pattern of distribution of D3 in the brain of rats strongly suggests that it is primarily expressed in neurons, but it is also present in astrocytes (183,184,185).
Very high levels of D3 activity and mRNA have been identified in hemangiomas in infants. In infants with very large tumors, the result is hypothyroidism, caused by very rapid deiodination of T4 and T3 (162). This syndrome, termed consumptive hypothyroidism, has also been described in an adult with a large hepatic hemangiopericytoma (186).
D3 activity has also been detected in the retina in rat fetuses and, in lesser amounts, in adults (187). In Xenopus laevis, the localized expression of D3 in the cells of the marginal zone of the retina accounts for the asymmetric growth of the retina (188). As noted above, D3 is highly expressed in the skin of adult rats (43,189), and in them skin contains more reverse T3 than any other tissue, suggesting that the high levels of D3 activity in homogenates of skin accurately reflect the activity of this enzyme in vivo (189). In this regard, T4 applied to normal human skin is largely converted to reverse T3 (190).
Large amounts of D3 are present in the placenta of rats, guinea pigs, and humans, and it is by far the predominant deiodinase present in this tissue (176,191,192,193,194). High levels of D3 are also present in the uterus of pregnant rats, initially in decidual cells and later in the single-cell layer of the epithelium (195). The levels of D3 are highest at the implantation site, nearly double the highest values obtained for placental tissue (196). In humans, the highest levels of D3 are in the endometrium (Fig. 7.7), and it is also found in fetal skin, tracheal and bronchiolar epithelium, mesothelium, and intestinal epithelium (197).
FIGURE 7.7. Type 3 deiodinase (D3) activity in the uterus and its myometrial and endometrial components. The endometrial (Endo) and myometrial (Myo) layers of the uterus from two nonpregnant women were dissected and assayed for D3 activity in the presence of 1 mM propylthiouracil. The values shown are maximum velocity, as determined by Lineweaver-Burke analysis.
Regulation of D3 Synthesis
Parallels between D3 activity and thyroid status have been demonstrated in several species, although the underlying molecular mechanisms remain obscure. In Xenopus laevis tadpoles, administration of T3 before the climax of metamorphosis results in a rapid and marked increase in D3 activity (39). In rats, D3 activity in the central nervous system is increased by thyroid hormone administration and decreased by hypothyroidism (164). In in situ hybridization histochemical studies, D3 gene expression within the central nervous system increased 4- to 50-fold in rats made thyrotoxic, with the greatest increase in the cerebellum. Conversely, D3 mRNA is not detectable in Northern blots of brain tissue of hypothyroid rats (174). Whether the dramatic increase in D3 mRNA in rats given T3 is due to increases in gene transcription, mRNA stabilization, or a combination of these factors is not known. In X. laevis, this T3 effect is not blocked by inhibition of protein synthesis. The promoter regions of the Dio3 gene are stimulated by T3, but the magnitude of this stimulation is modest as compared with the effect of T3 on D3 activity. Regulation of D3 activity by thyroid hormone has also been demonstrated in cultured astroglial cells. In these cells, the addition of 10 nM T3 (or T4) to the culture medium caused a slow increase in D3 activity, which reached a plateau in 48 hours (198).
D3 is expressed in the uterus, and the content increases during pregnancy. In rats, uterine D3 activity increases immediately after implantation, or artificial decidualization of the uterus in pseudopregnant rats, whereas the increases in activity are minimal in the nondecidualized uterine horn in the latter rats. In spontaneously cycling female rats, D3 activity was three to eight times higher during estrus, as compared with diestrus. Furthermore, the uterine levels of D3 activity were synergistically increased in ovariectomized rats given estradiol and progesterone in various combinations. Thus, estradiol and progesterone regulate thyroid hormone metabolism in the uterus, and the implantation process is a potent stimulus for the induction of D3 activity in this organ.
Most critically ill patients have low serum T3 and high serum reverse T3 concentrations (see section on nonthyroidal illness in Chapter 11). In patients with multiple organ system failure and other serious illnesses who died, hepatic D1 activity was low, and hepatic and skeletal muscle D3 activity was high (61). These findings suggest that T4 and T3 metabolism is altered in tissue-specific ways in illness, particularly with respect to reducing T3 formation or increasing T3 degradation. Similarly, D3 activity was increased in cardiac muscle of rats with cardiac hypertrophy and failure (199); among these rats, right ventricular D3 activity was significantly higher in those animals in which hypertrophy progressed to heart failure, as compared with the animals in which it did not. The induction of D3 in cardiac muscle would be expected to result in reduced intracellular concentrations of T3, which might reduce cardiac work and therefore help maintain cardiac compensation.
Extracellular Receptor Kinase–Activated Pathways
In cultured rat astroglial cells, factors that alter cellular processes through signaling cascades originating at the plasma membrane increase D3 activity. For example, D3 activity increases markedly and rapidly after exposure of the cells to 12-O -tetradecanoyl phorbol-13-acetate (TPA), fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and cyclic AMP analogues (200). The stimulatory effects of TPA and fibroblast growth factor on D3 mRNA and activity appear to be mediated at least partially by activation of the MEK/ERK signaling cascade (201).
NONDEIODINATIVE METABOLISM OF IODOTHYRONINES
Iodothyronines are mostly metabolized by deiodination. There are other pathways of metabolism, such as ether bond cleavage [mainly of T4 in leukocytes (202)], deamination and decarboxylation of the alanine side chain (203,204), sulfoconjugation mediated by cytosolic phenol sulfotransferases in several tissues (205), and glucuronidation and O-methylation, which renders the products more hydrophilic and thereby facilitates their excretion by bile, feces, and urine (206).
Sulfoconjugation is the most important alternative iodothyronine metabolic pathway. The sulfotransferases of liver are normally involved in inactivation and detoxification reactions, with a preference for lipophilic substrates (207,208). Sulfation of iodothyronines facilitates rapid inner ring deiodination by D1 but not by D3. All iodothyronines except reverse T3, the preferred substrate of D1, are sulfated to some extent (205).
Iodothyronines with two iodine atoms at the phenolic ring are preferentially conjugated with glucuronic acid, whereas iodothyronines that contain only one iodine atom in the phenolic ring are sulfated with the following preference: 3′-T1 = 3,3′-T2 > T3 > rT3 > T4 (166,205,207,209,210,211). T3 can be sulfated by human cytosolic liver phenol sulfotransferase (EC 184.108.40.206), with Km values in the 100 mM range, considerably greater than that for D1 (212). Biliary and urinary excretion of iodothyronine sulfates is a minor route for thyroid hormone elimination in humans. Considerable amounts of iodothyronine sulfates are detectable in plasma and bile after inhibition of D1 by PTU in rats (205,213,214). Moreover, at a high substrate concentration (> 1 mM) in vitro, metabolism proceeds mainly by sulfation, whereas at lower concentrations (< 0.1 mM), sulfation is the rate-limiting step; no sulfates accumulate because of the rapid deiodination of the sulfated iodothyronines. Thus far, the cellular capacity for conjugation seems to be unsaturable, in contrast to enzymic monodeiodination (29,166,209). In human hepatocarcinoma cells (HepG2 cells), inner ring deiodination of T3 is reduced, due to deficient T3 sulfation, which appears to be an obligatory step before deiodination of T3 (215).
Rats with selenium deficiency have high serum T3 sulfate concentrations and increased enterohepatic cycling of T3 sulfate, and the serum half-life of T3 is prolonged, as compared with normal rats (216). Under these conditions, sulfoconjugation might lead to greater availability of the active hormone. Sulfoconjugates of T4, T3, rT3, and 3,3′-T2 have been identified in human serum and amniotic fluid by specific radioimmunoassays (217,218,219,220,221). These conjugates are normal components of maternal and fetal serum and amniotic fluid. Their production and metabolism are regulated by the hormonal state, nonthyroidal illness, and drugs that inhibit deiodinase activity; sulfoconjugates of maternal origin may contribute to fetal thyroid economy.
PHYSIOLOGIC ROLES OF THE SELENODEIODINASES
D2 and Regulation of Thyrotropin Secretion
The main secretory product of the thyroid gland is T4, and serum T4 is a more important regulator of TSH secretion than serum T3. Because T3 is the biologically active thyroid hormone, T4 sensing must be preceded by its conversion to T3. The first evidence that there was a PTU-insensitive pathway for T4 to T3 conversion was that T4 very rapidly (within 30 minutes) inhibited TSH secretion in rats with hypothyroidism, and the inhibitory effect was not blocked by PTU (24,222,223,224). Subsequent studies in which combinations of 125I-T4 and 131I-T3 were injected revealed 125I-T3 bound to thyroid hormone receptors in the nuclei of pituitary cells within 15 minutes after injection of 125I-T4. This could not be explained by accumulation of 125I-T3 from serum, and was not inhibited by pretreatment with PTU (21,24). Pretreatment with iopanoic acid blocked both the generation of pituitary nuclear 125I-T3 and the biologic effect of T4 on TSH release (225).
The presence of D2 can account for the requirement for physiologic concentrations of both T4 and T3 for normal secretion of TSH. This requirement can account for the increase in TSH secretion that occurs in the early stages of iodine deficiency, when only T4, but not T3, production is decreased (226). Furthermore, normal serum concentrations of both T3 and T4 are required to suppress TRH mRNA production in the paraventricular nucleus of the hypothalamus and to normalize serum TSH concentrations in thyroidectomized rats (227,228,229). However, no D2 activity is present in this region of the hypothalamus; it is instead concentrated in the arcuate nucleus and median eminence (112,230). Subsequent in situ hybridization studies revealed that D2 is localized in the tanycytes (111,113,114). These specialized ependymal cells have their cell bodies in the inferior portion of the third ventricle, and they are probably where T4 is converted to T3, providing T3 that is released into the hypothalamic-pituitary portal system and carried to the thyrotrophs of the pituitary to regulate TSH secretion.
The thyroid secretes T4 and T3 in a proportion determined by the T4/T3 ratio in thyroglobulin (15/1 in humans), as modified by the minimal thyroidal conversion of T4 to T3 (231). Thus, the prohormone T4 is the major secreted iodothyronine in iodine-sufficient subjects; the molar ratio of secreted T4 to T3 is about 11 to 1 due to intrathyroidal T4 to T3 conversion via D1 and D2 (116,232,233). As noted above, most T3 production occurs in various extrathyroidal tissues via 5′-deiodination of T4 catalyzed by D1 and D2. The serum concentrations of free T4 and T3 are constant, but the concentrations of T4 and T3 in cells vary according the amounts of each hormone that are transported or diffuse into the cells, and the type and activity of the deiodinases in the cells. These deiodinases can increase (D2) or decrease (D3) the intracellular concentrations of T3 and consequently the nuclear content of thyroid receptor-T3 complexes independently of the serum T4 and T3 concentrations. As a result, the impact of T4 and T3 in serum on thyroid hormone action varies in different tissues. In liver and kidney, for example, the saturation of the thyroid receptors is approximately 50%, whereas in the central nervous system it is close to 95%. Lastly, tissue T3 concentrations change throughout development, partially as a result of changes in the activity of D2 and D3. The deiodinases also modulate the thyroid status of individual tissues in response to iodine deficiency, hypothyroidism, and thyrotoxicosis. Cells lacking the capacity to alter the rate of deiodination of T4 and T3 are the most affected, because their thyroid status will be determined primarily by the serum free T3 concentration. On the other hand, in cells expressing D2 or D3, changes in the activity of these enzymes mitigate fluctuations in serum free T4 and T3concentrations, constituting a potent mechanism for maintaining thyroid homeostasis.
The relative contributions of the two sources of T3, thyroid secretion and extrathyroidal deiodination of T4, can be quantified by determining the T4 to T3conversion rate, which is, on average, about 35% to 40% in normal humans (60). Hence, with a normal T4 production rate of approximately 110 nmol/day (85 µg/day), approximately 40 nmol (25 µg) of T3 are produced by peripheral deiodination of T3 and the remaining 10 nmol (6 µg) are released by the thyroid (Fig. 7.8).
FIGURE 7.8. Daily triiodothyronine (T3) production in humans and rats. The dotted lines in the cylinder representing human extrathyroidal production reflect the uncertainty about the contributions of type 1 deiodinase (D1) and type 2 deiodinase (D2) to this pool. Values given are based on studies cited in the text. Estimates for rats are normalized to 100 g body weight. To convert nmol to µg, multiply by 0.65, and to convert pmol to µg, multiply by 0.00065.
Both D1 and D2 contribute to extrathyroidal production of T3, but assessing the contribution of the two deiodinases in vivo is difficult. In patients with primary hypothyroidism receiving constant doses of T4, administration of PTU in a dose of 1000 mg daily for 7 to 8 days caused a 20% to 30% decrease in serum T3concentrations (234,235). In another study, conversion of radiolabeled T4 to T3 in serum was not reduced in patients given 1200 mg/day of PTU (236). The results of these studies suggest that D1-catalyzed T3 production is not a major component of extrathyroidal T3 production in normal humans. However, in patients with thyrotoxicosis, the contribution from D1 is higher, about 50% (50), due to an increase in D1 activity. Fractional conversion of T4 to T3 is increased when serum T4 concentrations are low, indicative of increased D2 activity, because D1-catalyzed T3 production is decreased (50,237,238,239). It is difficult to define which compartments or tissues contribute the most to extrathyroidal T3 production in humans (240); depending on the assumptions used, one can obtain estimates suggesting that as much as 81% or as little as 15% of T3 derives from rapidly equilibrating (D1-containing) tissues, with the remainder coming from slowly equilibrating (D2-containing) tissues.
The relative contributions of the D1 and D2 pathways to whole-body T3 production can be assessed more accurately in rats. In normal rats treated chronically with high doses of PTU to inhibit D1 activity, the T4 to T3 conversion rate is reduced by 50% (241). Accordingly, in T4-treated thyroidectomized rats, treatment with PTU results in a 50% decrease in serum T3 concentrations (223). The results of compartmental analyses of T4 to T3 conversion rates in rats are similar, assuming that T4 to T3 conversion in the rapidly equilibrating pool occurs via D1 and more delayed conversion via D2 (242). Taken together, these data indicate that D1 catalyzes about half of the daily extrathyroidal T3 production from T4 in rats. This estimate of 50% in rats is clearly higher than the estimate of approximately 25% in humans from the above-mentioned PTU studies (Fig. 7.8).
There are several implications of the above calculations. Based on the data available, D2-catalyzed 5′-deiodination of T4 appears to be a more important source of T3 than does D1-catalyzed 5′-deiodination of T4 in humans. Until recently, D2 activity was believed to be restricted mainly to the central nervous system and pituitary. The identification of D2 mRNA and activity in skeletal muscle and heart suggests a more important role for D2 in T3 production (26,243). The fact that only approximately 20% of serum T3 comes from thyroidal secretion, as opposed to about 40% in rats, also indicates a need for more widespread T4 to T3 conversion in humans.
Intracellular Triiodothyronine Homeostasis
Serum T3 equilibrates rapidly with most tissues. Both T4 and T3 are carried across the plasma membrane by stereospecific energy-dependent transporters (244,245), and they may also diffuse into cells. At equilibrium, one can estimate the nuclear T3 derived from serum T3 from the ratio of nuclear to serum T3 and the serum T3 concentration. Measurements of the maximum binding capacity of the thyroid receptors for T3 allow calculation of the degree of saturation of the receptors, which is normally 40% to 50% in most tissues (246). Thus, changes in serum T3 concentrations during thyrotoxicosis or hypothyroidism are mirrored by changes in the occupancy of the thyroid receptors in those tissues, and it is the latter that determines the intensity of the biologic actions of thyroid hormone. However, in selected tissues, especially pituitary gland, brain, and brown adipose tissue, additional T3 is provided by intracellular T4 to T3 conversion (24,247). This has been termed T3 (T4) to differentiate it from T3 (T3), the nuclear T3 derived directly from serum T3. These tissues contain D2, and in them the T3 generated by D2-catalyzed T4 deiodination supplements that from serum as though it were derived from a kinetically different pool. As a result, thyroid receptor occupancy by T3 is much higher (70%–90%), and 50% to 80% of this receptor-bound T3 is T3 (T4) (21,23,248).
These differences have been confirmed using constant infusions of radiolabeled T3 and T4 (249,250,251,252) and direct quantitation of nuclear T3 (253). Other tissues (e.g., liver, kidney) in which serum T3 is the only source of nuclear T3 contain mostly D1. As discussed above, confocal microscopic studies of transiently expressed D2 suggest that it is located in the endoplasmic reticulum in the perinuclear region, and T3 formed in this region might have preferential access to the nucleus. D1, however, is distributed in the periphery of the cell, typical of a plasma membrane protein. The rapid exit of T3 from D1-containing tissues and its retention in D2-containing tissues explain the three- to four-fold higher ratio of nuclear to cytoplasmic free T3 in brain than in liver, kidney, or heart (254). The consequence of the presence of D2 is that the impact of changes in T4 production on cellular T3 content can be dampened at a prereceptor level by compensatory alterations in D2 activity.
Iodine Deficiency and Hypothyroidism
Iodine availability can be rate limiting for thyroidal T4 and T3 production, and multiple thyroidal and extrathyroidal mechanisms have evolved to mitigate the consequences of iodine deficiency (see the section on thyroid iodine transport in Chapter 4 and the section on iodine deficiency in Chapter 11) (255). Accordingly, in iodine-deficient rats, growth, O2 consumption, and thermal homeostasis are similar to those of normal rats, despite approximately 10-fold higher serum TSH concentrations and very low serum T4 concentrations (256,257). However, if iodine deficiency is severe and prolonged, signs of hypothyroidism do eventually develop, with reduced O2 consumption and reduced activity of T3-dependent enzymes (258,259,260). It is difficult to distinguish between compensated iodine deficiency and hypothyroidism, except by measurements of T3 actions, because serum TSH concentrations are high at all stages.
Iodine deficiency results in a series of physiologic adaptations in the hypothalamic–pituitary–thyroid axis, similar to those that occur in hypothyroidism, which maintain serum and tissue T3 concentrations in the normal range, delaying the onset of hypothyroidism. The earliest thyroidal adaptation is a decrease in diiodotyrosine formation, with a consequent decrease in thyroidal T4 synthesis, whereas thyroidal T3 synthesis is maintained (226,261). TSH se cretion increases as serum T4 concentrations decrease, increasing thyroidal iodide transport and the subsequent steps of thyroid hormone production (226,261). This stimulation helps to maintain thyroid hormone production, but with continuing iodine deficiency the ratio of diiodotyrosine to monoiodotyrosine in thyroglobulin decreases by approximately 3-fold and the ratio of T4 to T3 decreases by approximately 25-fold; the latter is due to a decrease in thyroidal T4content, not to an increase in T3 content. The ratio of T4 to T3 in the serum of iodine- deficient subjects is also low, due to hypothyroxinemia, not an increase in serum T3 concentrations. Thus, the hallmarks of iodine deficiency, and the early phase of primary hypothyroidism, are low serum T4, normal serum T3, and high serum TSH concentrations (261,262,263).
The extrathyroidal adaptations to iodine deficiency or primary hypothyroidism are more complex and involve a high degree of tissue specificity. As noted above, the overall fractional conversion of T4 to T3 is increased in patients with hypothyroidism (237). This increase is due to an increase in D2 activity, and its effect is to maintain extrathyroidal T3 production (26,96,243). In rats, the fractional T4 to T3 conversion rate is not substantially changed by hypothyroidism, but extrathyroidal T3 production shifts from being relatively PTU sensitive (~50%) to being PTU insensitive (264), indicating that the relative contribution of D2-catalyzed T4 conversion to T3 increases substantially.
In tissues that express D2, the activity of this enzyme is increased during iodine deficiency or hypothyroidism, thus increasing the local fractional conversion of T4 to T3, and mitigating the decrease in serum T4 concentrations (22,73,265,266,267). This has been particularly well documented for the brain; because of the negative regulation of Dio2 gene transcription by thyroid hormone (268), D2 mRNA increases in iodine-deficient animals in all regions of the brain that contain D2, and D2 activity increases even more (267), similar to what occurs in hypothyroid rats (134). This increase in D2 activity is explained by the hypothyroxinemia of iodine deficiency per se acting at a posttranslational level, as noted above in the sections on regulation of D2 synthesis and degradation.
In hypothyroidism, there is not only an increase in fractional conversion of T4 to T3, but the clearance of T3 from the brain also is reduced. This is because D3 is a T3-dependent gene, and its activity correlates with thyroid status. In both fetal and adult rats with iodine deficiency, overall brain D3 activity decreases by 50% (73,164,174,269,270). However, in specific brain regions, such as the cerebral cortex, hippocampus, and cerebellum, D3 activity decreases by 80% to 90% (73). The consequences of the decrease in D3 activity are twofold. First, the residence time of T3 within the tissue is prolonged (271). Second, because T4 is also a substrate for D3, relatively more of it will be available within the tissue for conversion to T3 by D2. Particularly in tissues like the brain, in which the exchange of T3 with serum is slow and most of the T3 is generated in situ, it is likely that fluctuations in the rate of T3 degradation have a greater influence on tissue levels of T3 than in tissues in which cellular T3 is in more rapid equilibration with serum, such as the liver and kidney (272).
The increased fractional production of T3 from T4 by D2 combined with the prolonged residence time of T3 mitigates the effects of severe iodine deficiency and hypothyroidism. This has been demonstrated in mild to moderate hypothyroidism using tracer studies (273). These pre dictions were confirmed directly by measuring thyroid hormone concentrations in various regions of the CNS in iodine-deficient rats (265). As expected, tissue T4 concentrations were markedly decreased, whereas tissue T3 concentrations were reduced by only 50%. This illustrates the effectiveness of these compensatory mechanisms.
Thyroid hormone is critically important for the coordination of developmental processes in all vertebrate species. During embryogenesis, thyroid hormone acts primarily to promote differentiation and thus attenuate proliferation. As a result, either insufficient levels of T3 or premature exposure of the embryo to high T3 concentrations result in abnormal development (274). As an example, exposure of neonatal rats to excess thyroid hormone causes accelerated morphogenesis of pyramidal neurons and their dendritic spines in the cerebrum and a persistent reduction in total number of neurons (275). The best characterized action by which thyroid hormone influences developmental processes is via changes in gene expression initiated by the binding of T3 to its receptors (276,277). During development in experimental animals, two deiodinases (D3 and D2) exert the major control of T3 concentrations (43). As mentioned earlier, fetal serum T3 concentrations are very low, and during early development D3 is the predominant deiodinase expressed in most rat tissues, and its activity in these tissues is much higher than in adult rats. In human fetuses, D3 is also present in the liver, and its content decreases near the end of gestation (167). This pattern suggests that D3 plays a major role in preventing premature exposure of fetal tissues to T3.
Conversely, D2 is expressed in most mammalian tissues for a limited period of time during development. This suggests that there is a tissue-specific program of T3-dependent differentiation. This has been found during tadpole metamorphosis, and it likely occurs during neuronal and glial maturation and cochlear maturation in rats (39,110,278,279,280,281). Finally, D1 activity is generally lower during fetal development than at later stages of life (282). This also would result in lower serum T3 concentrations at that time of life.
Deiodinases in Mammalian Development
Precisely timed D2 expression is fundamental during critical periods of mammalian development. In rat brain, D2 increases rapidly after birth, reaching its highest level around day 28. It then declines, reaching adult levels by day 50 (165). The cochlea is among the organs most sensitive to thyroid deficiency, as is evident from the deafness that is associated with endemic cretinism. In rats, complete cochlear maturation and the onset of auditory function require the presence of T3 between the late embryonic stage and the second postnatal week. So far, little is known about the mechanisms that control this temporal regulation. Analysis of cochlear homogenates from 2- to 8-day old rat pups revealed a striking peak of D2 activity peak at about 7 days, followed by an abrupt decline by day 10, a few days before the onset of hearing (110). Relative to serum, cochlear tissue has a high ratio of T3 to T4, supporting a role for D2 in increasing local T3 concentrations. D2 mRNA is localized in connective tissue near the region where dendritic and axonal projections of the cochlear nerve connect with the hair cells. D2 expression is complementary to, rather than coincident with, that of the βsubtype of the thyroid receptor, suggesting a paracrine rather than endocrine mode of signaling in cochlear tissue. D2-deficient mice have defective auditory function, retarded differentiation of the cochlear inner sulcus and sensory epithelium, and deformity of the tectorial membrane. The similarity of this phenotype to that caused by thyroid receptor mutations suggests that D2 controls the T3 signal that activates thyroid receptors in the cochlea (283).
In humans, the capacity to synthesize thyroid hormones does not appear until 10 to 12 weeks of gestation (see Chapter 74). However, human fetuses have thyroid receptors containing T3 at an earlier time (284); this T3 must come from the maternal circulation, via the placenta and the coelomic and amniotic fluids (285). At 6 to 12 weeks' gestation, the average total T4 concentrations are 11.3, 0.07, and 0.002 µg/dL (146, 0.96, and 0.02 nM), respectively, in maternal serum, coelomic fluid, and amniotic fluid, suggesting a marked gradient of T4 from mother to fetus. The gradient for reverse T3 is in the opposite direction, its concentrations being 3.8 and 15 times higher in the coelomic fluid and amniotic fluid, respectively, as compared with maternal serum (285). Also during the second and third trimesters, there are marked maternal-to-fetal gradients of free T4 and T3 (286,287).
D2 and D3 activity appear in fetal tissues at midgestation, whereas D1 is not evident until later (44). Accordingly, fetal serum T3 concentrations are very low before 30 weeks; thereafter they increase slightly, due to an increase in D1 activity in fetal tissues. The serum concentrations of sulfated iodothyronine concentrations are high in fetuses; although T3 sulfate does not bind to thyroid receptors, local desulfation, if it occurred, would provide a local source of T3 in fetal tissues (217,220,288,289). The pattern of circulating iodothyronines in the fetus, which is characterized by low serum T3 and high reverse T3concentrations, is due to the combination of high D3 activity and low D1 activity in fetal tissues throughout most of gestation.
Triiodothyronine Modulation of Placental Thyroid Hormone Transfer
The placenta is the pathway for maternal-fetal thyroid hormone transfer, and therefore can be an important determinant of the thyroid status of the fetus. Placental D3 activity increases with gestational age in rats and humans (176,192,290,291). In the first trimester, when the placenta and the surface area available for transfer are small, the activity of D3 per unit of placental tissue is high. At term, the activity per unit is lower, but because the placenta is much larger total placental D3 activity is higher. In rats, unlike humans, placental D3 activity increases about twofold from embryonic day 14 until day 16 or 17, after which it decreases (176,292,293). As mentioned above, placenta also contains D2; however, at all gestational ages, placental D3 activity is approximately 200-fold higher than is that of D2. In placental tissue, there is no direct correlation between D2 activity and mRNA levels, and although D3 activity is always higher than D2 activity, their mRNA levels are comparable (291).
The cellular localization of D2 and D3 in placental tissue also differs. D2 activity is higher in the chorionic and decidual membranes of the placenta than in the amniotic membranes, whereas D3 is found mostly in trophoblasts (194). However, given the low levels of D2 at all gestational ages, fluctuations in D2 activity are not likely to have a major effect on fetal serum T3 concentrations.
The physiologic consequence of the high placental D3 activity is clear. Placental tissue actively deiodinates T4 to reverse T3 and T3 to 3,3′-diiodothyronine, some of which is further deiodinated to 3′-monoiodothyronine (191). In isolated perfused human placental lobules, little of the T4 added to the maternal side appears on the fetal side. In contrast, reverse T3 concentrations rise progressively on both sides. Addition of the deiodinase inhibitor iopanoic acid to the maternal perfusate results in an increase in T4 and a decrease in reverse T3 on the fetal side, providing direct evidence that human placental D3 is a major factor controlling transfer of maternal T4 to the fetus (294).
The uterus of pregnant rats also contains high levels of D3 activity, initially in decidual cells and later in the single-cell layer of the epithelium (195). D3 activity is highest at the implantation site, and the activity there is almost double the highest value found in the placenta. These changes precede the appearance of thyroid receptor mRNA (295). Throughout gestation, D3 activity remains higher in the uterus than in the placenta, and is 10 times higher than in the entire fetus. D3 activity has also been detected in amniotic fluid (195). High uterine and placental D3 activity also protects against high T3concentrations, which can induce structural abnormalities in the cephalic and branchial arches (296).
The presence of D3 in the endometrium and placenta villae as well as every epithelial surface of the human fetus constitutes a barrier to inappropriate transfer of maternal T4 and T3 to the fetus (197). This barrier is so potent that instillation of 700 µg of T4 into amniotic fluid at term results in little increase in umbilical cord serum T3 concentrations of infants born 24 hours later (297). The high level of D3 activity in the uterus and placenta contributes to the need for more T4 in women with hypothyroidism when they are pregnant (298).
Paradoxically, despite inactivation of T4 and T3 by the uterus and placenta, neonates with congenital hypothyroidism often have little evidence of hypothyroidism at birth. Among infants with a complete defect in thyroid iodide organification, cord serum T4 concentrations are 20% to 50% of those in normal infants, indicating there is substantial transplacental passage of T4. The concentrations decrease rapidly after birth in these infants (299). These results indicate that a steep maternal-fetal gradient of T4 overcomes the placental barrier, permitting maternal T4 to enter the fetal circulation.
THE DEIODINASES IN HUMAN PATHOPHYSIOLOGY
Extrathyroidal conversion of T4 to T3 is decreased in patients with virtually all nonthyroidal illness, as discussed above and in the section on nonthyroidal illness in Chapter 11.
As noted above, high levels of D3 are expressed in hemangiomas. If these tumors are sufficiently large, the rate of T4 and T3 deiodination can exceed thyroidal production of the hormones, even in the presence of intense TSH stimulation. The first patient documented to have this condition was a 3-month-old infant who had severe hypothyroidism, with high serum TSH, undetectable T4 and T3, and high reverse T3 concentrations. To reverse the hypothyroidism rapidly, the infant was given T3 and T4 intravenously in doses sufficient to reduce TSH secretion to normal. The respective daily doses were 96 µg of T3 and 50 µg of T4 (162), doses that would cause overt thyrotoxirosis in not only normal infants but also normal adults (300). Remarkably, even during the infusions, serum T3 concentrations barely reached the normal range, serum T4 was never detectable, and serum reverse T3 concentrations were very high, providing direct evidence of excessive inner ring deiodination. D3 activity was subsequently identified in the infant's hemangioma at levels eight times that in the placenta, and in situ hybridization localized the D3 mRNA to hemangioma cells. Other patients with this unique syndrome have now been reported (301).
The relationship between infantile hemangiomas and D3 expression is important because it identifies a cause of hypothyroidism at a critical age for neurologic development. Although large hepatic hemangiomas can be fatal, a substantial fraction of these infants survive with therapy and the natural propensity of these tumors to regress. If hypothyroid, these patients may require high doses of T4 in addition to therapy directed at their hemangiomas.
D3 Expression and the Requirement for Thyroxine in Normal Pregnancy
Most pregnant women with hypothyroidism need an increase in T4 dose of approximately 40% to maintain normal serum TSH concentrations (299,302). The need for an increase begins early in pregnancy and persists until delivery (303), and its existence indicates that T4 and T3 production must increase in normal pregnant women. The causes of this need for more T4 and T3 include an increase in uterine D3 activity and the presence of placental D3 activity, transfer of T4and T3 to the fetus, and the estrogen-induced increase in serum thyroxine-binding globulin concentrations.
Overexpression of Deiodinases and Excess Triiodothyronine Production
In patients with thyrotoxicosis, there is a disproportionate increase in the production rate of T3 and serum T3 concentrations, as compared with those of T4, by a factor of two (50). The human Dio1 gene promoter is T3-responsive, and D1 mRNA levels are increased in thyroid tissue and mononuclear leukocytes in patients with thyrotoxicosis (42,304,305). In these patients, PTU—which blocks D1, but not D2, activity—results in a more rapid acute decrease in serum T3concentrations than does methimazole, which has no effect of either D1 or D2 activity (50). These results indicate that D1-catalyzed T4 to T3 conversion is increased in patients with thyrotoxicosis (234,235,236). This has led to the recommendation that patients with severe thyrotoxicosis be treated with large doses of PTU or other drugs that block T4 conversion to T3 (see Chapter 45) (306,307,308,309).
In patients with Graves' thyrotoxicosis, thyroidal D2 mRNA is increased despite high serum T4 and T3 concentrations (116). This is presumably due to the effect of TSH receptor–stimulating antibodies to activate the cyclic AMP-dependent Dio2 gene, which overwhelms the negative transcriptional effect of T3 on this gene. Therefore, some of the relative excess of T3 production in patients with thyrotoxicosis is caused by an increase in thyroidal D2 activity (116).
Another consequence of thyroid overexpression of D2 is illustrated by three athyreotic patients with widespread follicular carcinoma of the thyroid in whom the ratio of T3 to T4 in serum was persistently high, in the absence of autonomous production of T3 by the tumor. In one patient, the tumor contained a high level of D2 activity, and resection of the tumor resulted in a normal T4 to T3 ratio in serum. In two other patients, treatment with T4 in doses sufficient to suppress TSH secretion was associated with high normal serum T3 and subnormal free T4 values (310). These findings indicate that increases in T4 conversion to T3, presumably caused by increased D2 activity, can alter serum T4 and T3 concentrations.
Many tissues contain deiodinase activity, and it is now clear that these enzymes are important determinants of thyroid hormone homeostasis in both health and disease. Two deiodinases catalyze the intracellular conversion of T4 to T3, and a third catalyzes intracellular conversion of T4 and T3 to biologically inactive products. Variations in the activity of these enzymes, as a result of thyroid and other disorders, can affect thyroid hormone availability and therefore thyroid hormone action, in many tissues.
This work was supported by National Institutes of Health Grants DK36256, DK44128, and DK58538.
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