Type 1 Iodothyronine Deiodinase Is a Sensitive Marker of Peripheral ...

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Endocrinology 146(3):1568 –1575 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2004-1392

Type 1 Iodothyronine Deiodinase Is a Sensitive Marker of Peripheral Thyroid Status in the Mouse Ann Marie Zavacki, Hao Ying, Marcelo A. Christoffolete, Goele Aerts, Edward So, John W. Harney, Sheue-yann Cheng, P. Reed Larsen, and Antonio C. Bianco Thyroid Section (A.M.Z., M.A.C., E.S., J.W.H., P.R.L., A.C.B.), Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women’s Hospital, Boston, Massachusetts 02115; Laboratory of Comparative Endocrinology (G.A.), Katholieke Universiteit Leuven, 3000 Leuven, Belgium; and Gene Regulation Section (H.Y., S.C.), Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892 Mice with one thyroid hormone receptor (TR) ␣-1 allele encoding a dominant negative mutant receptor (TR␣1PV/ⴙ) have persistently elevated serum T3 levels (1.9-fold above normal). They also have markedly increased hepatic type 1 iodothyronine deiodinase (D1) mRNA and enzyme activity (4- to 5-fold), whereas other hepatic T3-responsive genes, such as Spot14 and mitochondrial ␣-glycerol phosphate dehydrogenase (␣-GPD), are only 0.7-fold and 1.7-fold that of wild-type littermates (TR␣1ⴙ/ⴙ). To determine the cause of the disproportionate elevation of D1, TR␣1ⴙ/ⴙ and TR␣1PV/ⴙ mice were rendered hypothyroid and then treated with T3. Hypothyroidism decreased hepatic D1, Spot14, and ␣-GPD mRNA to similar levels in TR␣1ⴙ/ⴙ and TR␣1PV/ⴙ mice, whereas T3 administra-

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HE TYPE 1 IODOTHYRONINE deiodinase (D1) is a member of a group of selenoenzymes that metabolize thyroid hormone and thus modulate thyroid hormone action. Both D1 and the type 2 iodothyronine deiodinase (D2) can catalyze the conversion of T4 to the ligand for thyroid hormone receptor (TR), T3. Unlike D2, however, D1 can catalyze both activation of T4 by outer-ring deiodination and inactivation of T4 by inner-ring deiodination to produce rT3. Although D1 is thought to provide a modest fraction of the plasma T3 of euthyroid humans, it makes a more substantial contribution in patients with hyperthyroidism. D2 is important in the generation of intracellular T3, thus locally controlling thyroid status, and also has been recently appreciated to provide a significant fraction of the circulating T3 in humans. Another family member, the type 3 iodothyronine deiodinase (D3), can inactivate T4 and T3 by inner-ring deiodination, thus blocking thyroid hormone action (1). Given their crucial role in controlling the availability of the biologically active thyroid hormone, it is not surprising that the D1, D2, and D3 enzymes should also be regulated by thyroid hormones. For example, during iodine deficiency or hypothyroidism, plasma T4 is reduced and peripheral T3 production from T4 is sustained by up-regulation of D2 and First Published Online December 9, 2004 Abbreviations: D1, Type 1 iodothyronine deiodinase; D2, type 2 iodothyronine deiodinase; D3, type 3 iodothyronine deiodinase; DTT, dithiothreitol; ␣-GPD, ␣-glycerol phosphate dehydrogenase; MMI, methimazole; TR, thyroid hormone receptor. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

tion caused an approximately 175-fold elevation of D1 mRNA but only a 3- to 6-fold increases in Spot14 and ␣-GPD mRNAs. Interestingly, the hypothyroidism-induced increase in cerebrocortical type 2 iodothyronine deiodinase activity was 3 times greater in the TR␣1PV/ⴙ mice, and these mice had no T3-dependent induction of type 3 iodothyronine deiodinase. Thus, the marked responsiveness of hepatic D1 to T3 relative to other genes, such as Spot14 and ␣-GPD, explains the relatively large effect of the modest increase in serum T3 in the TR␣1PV/ⴙ mice, and TR␣ plays a key role in T3-dependent positive and negative regulation of the deiodinases in the cerebral cortex. (Endocrinology 146: 1568 –1575, 2005)

down-regulation of D1. Because D2 is a much more catalytically efficient enzyme [with a Michaelis-Menten constant (Km) ⬃1000 times lower than D1] and produces only T3 from T4, this results in an increase in the fractional conversion of T4 to T3. On the other hand, during hyperthyroidism, the efficiency of T4 deiodination to T3 is greatly reduced because D1, an enzyme that produces equimolar amounts of T3 and the metabolically inactive rT3, is increased, whereas D2 is reduced (1). During hypothyroidism, a reduction in D3 activity decreases the metabolic clearance rate of both T4 and T3, whereas the opposite is observed during hyperthyroidism (1). Accordingly, a series of T3- and T4-mediated transcriptional and posttranslational mechanisms regulating the deiodinases serve to maintain thyroid hormone homeostasis. It is well known that T3 binds to the ␣ and ␤ isoforms of TR (TR␣ and TR␤), which are in turn bound to specific DNA sequences known as thyroid hormone response elements. This permits positive or negative regulation of gene transcription by T3 (2). Thus, hepatic Dio1 is positively regulated by T3 at the transcriptional level in humans, mice, and rats (3–5). Additionally, Dio2 expression is down-regulated by its end product, T3, and it is potently negatively regulated at a posttranslational level in the presence of its substrate, T4, by selective proteolysis via the ubiquitin-proteasomal pathway. Furthermore, Dio3 expression is induced by the substrate it inactivates (T3), with both D3 enzyme activity and mRNA levels increasing with T3 treatment (1). An interesting model of disrupted thyroid hormone action, resulting in the dysregulation of the Dio1 gene, can be found in TR␣1PV/⫹ mice (6). These mice have a targeted replacement of one allele of TR␣ with a mutant receptor

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Zavacki et al. • D1 Is a Sensitive T3 Marker

containing a mutation identical to that found in the TR␤ gene in a family with thyroid hormone resistance, the PV kindred. A cytosine insertion at position 1180 of the mouse TR␣1 gene results in a frame shift that disrupts the last 17 amino acids associated with the activation function-2 (AF-2) region of TR␣. This mutant TR␣ is unable to bind T3, resulting in strong dominant negative activity that can inhibit TR␣ and TR␤ function in vitro (6). TR␣1PV/⫹ mice exhibit a mild resistance to thyroid hormone, with T3 and TSH levels both being slightly elevated, and are smaller in stature, have impaired fertility, and have increased premature mortality. Interestingly, although a wide range of T3 target genes in pituitary, cerebellum, and liver showed either no change or a slight increase in expression in response to the persistent 15% elevation in serum T3 in TR␣1PV/⫹ mice, in the original report, hepatic D1 mRNA levels were elevated more than 9-fold over littermate controls (TR␣1⫹/⫹) (6). If this modest increase in serum T3 of the TR␣1PV/⫹ mice is the sole explanation for the increased hepatic D1 expression, Dio1 would be the most sensitive indicator of peripheral thyroid status in the mouse genome currently known. To assess whether the modest increase in serum T3 is the only cause of the increased Dio1 expression in the TR␣1PV/⫹ mice, animals were rendered hypothyroid and subsequently treated with T3, and then expression of Dio1 and other T3responsive genes was compared. Our results indicate that the chronic modest elevation of serum T3 in the TR␣1PV/⫹ mice does result in a disproportionate increase in Dio1 expression relative to other hepatic T3-responsive genes and that a hyperresponsiveness of Dio1 to exogenous T3 is found in both the TR␣1PV/⫹ and another mouse strain. In addition, the cerebral cortex of the TR␣1PV/⫹ mice displayed enhanced expression of the negatively T3-regulated Dio2 gene under conditions of hypothyroidism, and T3-mediated induction of D3 activity in this tissue was lost, which is consistent with an important role for TR␣ in transducing the T3 responsiveness of the Dio2 and Dio3 genes in this tissue. Materials and Methods Animal treatment protocols Animals were maintained and experiments were performed according to protocols approved by the Animal Care and Use Committees of either Harvard Medical School (Boston, MA) or the National Institutes of Health (Bethesda, MD). The TR␣1PV/⫹ mice were generated as previously described, with chimeras being backcrossed into an National Institutes of Health Black Swiss background (6). C57BL/6J mice were obtained from Jackson Labs (Bar Harbor, ME). Mice were fed normal chow and were housed under a 12-hour light, 12-hour dark cycle at 22 C. For the TR␣1PV/⫹ experiments, male mice of 2– 4 months of age were used, with the TR␣1⫹/⫹ group consisting of normal male littermates. Mice were rendered hypothyroid by the addition of 0.1% methimazole (MMI) and 1% NaClO4 (Sigma, St. Louis, MO) in their drinking water for 21 d. As indicated, some animals from this group were injected ip with 5 ␮g T3 per mouse for 5 d (⬃60 times the estimated replacement dose of 3.5 ng/g䡠d) (7), on d 16 –20 of MMI/NaClO4 treatment. Mice were killed 24 h after their last T3 injection by exsanguination under anesthesia on d 21. Control groups received no treatment. TR␣1PV/⫹ mice weigh on average about 70% of the weight of TR␣1⫹/⫹ mice. To compensate for this, this experiment was repeated in its entirety, and mice were injected with 5 ␮g T3/20 g of body weight, with similar results being obtained. In separate experiments designed to evaluate the T3 response of Dio1

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relative to other hepatic T3-responsive genes in C57BL/6J mice, a similar protocol was followed. Two-month-old female C57BL/6J mice were injected ip with 2 ␮g T3 per mouse (⬃34 times the replacement dose) for 3 d on d 22–24 of their MMI/NaClO4 treatment and killed 24 h after the last T3 injection on d 25. The control group of mice was untreated and killed on d 26 of the experiment.

T3 and T4 measurements Serum T3 values were measured as described previously (8) with the following minor modifications: the standard curve was prepared by diluting a known amount of T3 into charcoal-stripped mouse serum (Sigma) and the primary anti-T3 antibody (rabbit D) was used at a 1:100,000 final concentration. Serum T4 was measured using the COATA-COUNT total T4 kit (DPC, Los Angeles, CA), following the manufacturer’s instructions, with a mouse T4 standard curve prepared in charcoal-stripped mouse serum.

Charcoal uptake An estimate of the free fraction of serum T3 was determined using a modification of previously described methods (9). Ten microliters of mouse serum were diluted into 0.5 ml of PBS (pH 7.4) containing approximately 7000 cpm of 125I-T3, with a specific activity of 1080 –1320 ␮Ci [(40.0 – 48.8 MBq)/␮g; NEN Life Science Products, Boston, MA)]. Samples were allowed to equilibrate for 45 min at room temperature and then transferred to an ice water bath for 15 min. Prechilled 0.0125% activated charcoal (0.5 ml; Sigma) solution in PBS was added, and samples were incubated on ice for an additional 15 min. Samples were spun in a Beckman J6 centrifuge at 2500 rpm for 15 min, and the charcoal-containing pellets were counted. Conditions were optimized such that approximately 30% of the tracer was bound to charcoal in sera from euthyroid control mice, and samples were assayed in duplicate for each mouse. To account for interassay variation, values were normalized to the average uptake of the control TR␣1⫹/⫹ mice in each assay (percent uptake from 22–28%, with ses not greater than 10%). An estimate of the free T3 (free T3 index) can be calculated by multiplying the total T3 concentration by the normalized T3 charcoal uptake.

D1, D2, and D3 assays Deiodinase assays were performed as described previously (10). Briefly, tissues were sonicated in buffer containing 0.1 m KPO4, 1 mm EDTA, 0.25 m sucrose, and 10 mm dithiothreitol (DTT). Protein concentrations were determined by Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA), and the following conditions were used. For D1 assays, 5–15 ␮g of protein (liver) or 15–50 ␮g of protein (kidney) was assayed with a final concentration of 10 mm DTT and 500 nm 125I-rT3. For D2 assays, 20 –125 ␮g of cerebral cortex protein was assayed with a final concentration of 20 mm DTT, 0.5 nm 125I-T4, and 1 mm propylthiouracil. For D3 assays, 20 –125 ␮g of cerebral cortex protein was assayed with a final concentration of 10 mm DTT, 10 nm 125I-T3, and 1 mm propylthiouracil.

Real-time PCR RNA was extracted from liver using Trizol Reagent (Invitrogen, Carlsbad CA), visualized by electrophoresis to ensure integrity, and used to synthesize cDNA using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) with an oligo-dT primer. mRNA levels were measured by quantitative real-time PCR using the QuantiTect SYBR Green PCR kit (Bio-Rad) in an I-Cycler (Bio-Rad). Standard curves (a 5-point serial dilution of mixed experimental and control group cDNAs) were analyzed in each assay and used as calibrators to determine the relative expression of each gene within the assay when measured in the exponential phase of the amplification curve. All values were normalized using an internal control of ␤-actin mRNA. Similar results were obtained when cyclophilin mRNA was used as an internal control (data not shown). Primers were designed using Beacon Designer (Premier Biosoft International, Palo Alto, CA) and synthesized by Invitrogen. The primers were as follows: 5⬘-CCACCTTCTTCAGCATCC-3⬘ and 5⬘-AGTCATCTACGAGTCTCTTG-3⬘ amplify the mouse D1 cDNA; 5⬘-TGCTAACGAAACGCTATCC-3⬘ and 5⬘-TTCTACACAGTGCTCTTGG-3⬘ amplify mouse Spot14 cDNA; 5⬘-GTGTGCGAT-

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ACCTCCAGAAG-3⬘ and 5⬘-GTTGTGTTGTCCGTCATAGTAG-3⬘ amplify the mouse ␣-glycerol phosphate dehydrogenase (␣-GPD) cDNA; 5⬘CTTCTCTACCACCACCTTC-3⬘ and 5⬘-CATCTTCACCCAGTTTAACC-3⬘ amplify the mouse D2 cDNA; and 5⬘-CACACCCGCCACCAGTTC-3⬘ and 5⬘-GCCACACGCAGCTCATTG-3⬘ amplify the mouse ␤-actin gene.

Statistical analysis One-way ANOVA with a Newman-Keuls posttest was used to determine significant differences between groups and was performed using Prism 3.0 (GraphPad Software, San Diego, CA). When only two groups were analyzed, statistical significance was determined by a twotailed Student’s t test, using Prism 3.0 software. Critical P values are as indicated but are not shown for all statistically different results.

Results TR␣1PV/⫹ mice have elevated serum T3 and impaired serum T3 clearance

T4 levels were not different between the two control groups of mice, as reported previously; although the control group of TR␣1PV/⫹ mice had serum T3 levels 1.9-fold those of the TR␣1⫹/⫹ control group (Table 1). This difference is greater than previously reported and may reflect the use of a more specific RIA method. To determine whether the higher serum T3 levels in the TR␣1PV/⫹ mice could be due to differences in serum thyroid hormone binding proteins, charcoal T3 uptake assays were performed. The charcoal uptakes, which provide an estimate of the free fraction of T3, were 1.0 ⫾ 0.02 and 1.28 ⫾ 0.02 for the TR␣1⫹/⫹ and TR␣1PV/⫹ mice, respectively (values ⫽ mean ⫾ se, n ⫽ 8 –9 mice/group; P ⬍ 0.01 by Student’s t test). Thus, the TR␣1PV/⫹ mice have a higher free T3 fraction (i.e. lower T3 binding to serum proteins) than their littermate controls. Taking this difference into account further magnifies the difference in the serum T3 levels of the TR␣1PV/⫹ vs. TR␣1⫹/⫹ mice, with free T3 estimates being approximately 3 times higher in the TR␣1PV/⫹ animals. To determine whether the markedly elevated D1 mRNA previously observed in TR␣1PV/⫹ mice could be explained simply on the basis of a greater induction due to the increase in serum T3, TR␣1PV/⫹ mice and TR␣1⫹/⫹ littermates were rendered hypothyroid and then treated with T3 (Fig. 1). Both TR␣1PV/⫹ and TR␣1⫹/⫹ hypothyroid animals had an undetectable serum T4 and similarly decreased T3 values, indicating that the protocol for induction of hypothyroidism was effective (Table 1). In the T3-treated group, serum T3 concentrations were close to those of the TR␣1⫹/⫹ control group 24 h after the last T3 injection, as previously reported for this protocol (5). This would be expected given that the half-life of T3 in mouse serum is approximately 2 h (11). In the T3-

FIG. 1. Schematic of different treatments of TR␣1⫹/⫹ and TR␣1PV/⫹ mice. Mice heterozygous for the TR␣ PV mutation (TR␣1PV/⫹) or normal littermates (TR␣1⫹/⫹) were treated with 0.1% MMI and 1% NaClO4 in their drinking water for a total of 26 d (hypothyroid), not treated (control), or treated with 0.1% MMI and 1% NaClO4 in their drinking water for 21 d and injected with 5 ␮g of T3 at 24-h intervals on d 16 –20 (T3 treated). Twenty-four hours after the last T3 injection, on d 21, mice were killed by exsanguination, and tissues were collected.

treated TR␣1PV/⫹ group of mice, however, serum T3 levels were significantly higher, being 8.5 times greater than those of the T3-treated TR␣1⫹/⫹ group 24 h after their last T3 injection, which suggests that these mice have a defect in T3 clearance (Table 1). Because TR␣1PV/⫹ mice weigh only 70% of the weight of TR␣1⫹/⫹ mice (6), this experiment was repeated by injecting 5 ␮g T3/20 g of body weight for 5 d to control for the size difference between the two groups of mice. Similar results were obtained, with serum T3 concentrations of the T3-treated TR␣1PV/⫹ group being 5.3 times the concentrations of the T3-treated TR␣1⫹/⫹ group 24 h after the last T3 injection. D1 activity is elevated in TR␣1PV/⫹ mice and retains normal T3 responsiveness in liver and kidney

Liver D1 activity was 4.8-fold higher in the TR␣1PV/⫹ mice (P ⬍ 0.05 by ANOVA) than in TR␣1⫹/⫹ controls (Fig. 2A). Basal D1 activity decreases in both hypothyroid TR␣1⫹/⫹ and TR␣1PV/⫹ mice, whereas hepatic D1 levels are greatly increased to 369 and 246 pmol/min/mg, respectively, with T3 treatment, indicating normal T3 responsiveness of Dio1 in both groups of mice. When this experiment was repeated with the amount of T3 injected being normalized for differences in body weight between the TR␣1⫹/⫹ and TR␣1PV/⫹ mice, similar results were obtained (data not shown). D1 activity was also measured in kidneys from the TR␣1⫹/⫹ and TR␣1PV/⫹ mice (Fig. 2B). D1 activity was 3.2 pmol/min/mg in the control group of the TR␣1⫹/⫹ mice and 8.8 pmol/min/mg in TR␣1PV/⫹ mice, although this 2.8-fold

TABLE 1. Serum T3 and T4 concentrations in hypothyroid, control, and T3-treated TR␣1⫹/⫹ and TR␣1PV/⫹ mice TR␣1⫹/⫹

T3 (ng/ml) T4 (␮g/dl)

TR␣1PV/⫹

Hypothyroid

Control

T3 treated

Hypothyroid

Control

T3 treated

0.25 ⫾ 0.06 ⬍0.05c

0.63 ⫾ 0.05 1.5 ⫾ 0.35

0.53 ⫾ 0.06 ⬍0.05

0.26 ⫾ 0.10 ⬍0.05

1.2 ⫾ 0.17a 1.8 ⫾ 0.31

4.5 ⫾ 0.17b ⬍0.05

Values are the mean ⫾ SE of three to five mice/group treated as indicated in Fig. 1. Both T3-treated groups of mice were killed 24 h after the last of five daily ip T3 injections. a P ⬍ 0.01 compared with control TR␣1⫹/⫹ by ANOVA. b P ⬍ 0.01 compared with T3-treated TR␣1⫹/⫹ by ANOVA. c ⬍ 0.05 ⫽ below the detectable limits of the assay.



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FIG. 2. D1 activity in liver and kidney of hypothyroid (hypo), control (ctrl), and T3-treated (T3) TR␣1⫹/⫹ and TR␣1PV/⫹ mice. TR␣1⫹/⫹ or TR␣1PV/⫹ mice were treated as indicated in Fig. 1, and D1 enzyme activity in (A) liver or (B) kidney was measured. Results are the mean of three to five mice ⫾ SE. P values shown were determined using ANOVA.

difference was not statistically significant. Furthermore, kidney D1 activity did not decrease significantly in the hypothyroid TR␣1⫹/⫹ or TR␣1PV/⫹ mice. Kidney D1 activity was greatly increased to 44.9 and 54.3 pmol/min/mg in both the TR␣1⫹/⫹ and TR␣1PV/⫹ mice, respectively, with T3 treatment. Dio1 gene expression is increased to a greater extent with T3 treatment than other hepatic T3-responsive genes

Liver D1 mRNA levels were 4.1-fold higher in untreated TR␣1PV/⫹ mice when compared with TR␣1⫹/⫹ littermates, whereas Spot14 levels were not significantly different (0.7fold), and the levels of mitochondrial ␣-GPD were only slightly higher (1.7-fold; Fig. 3). Hypothyroidism markedly decreased D1, Spot14, and ␣-GPD mRNA levels in both TR␣1PV/⫹ and TR␣1⫹/⫹ mice, and T3 treatment increased D1 mRNA levels approximately 150- to 200-fold relative to hypothyroid levels, whereas Spot14 and ␣-GPD levels were only increased 3- to 6-fold. To further evaluate the intrinsic responsiveness of the mouse Dio1 gene to T3 in a different mouse strain, similar

FIG. 3. Hepatic D1, Spot14, and ␣-GPD mRNA levels of hypothyroid (hypo), control (ctrl), and T3-treated (T3) TR␣1⫹/⫹ and TR␣1PV/⫹ mice. RNA was extracted from the livers of TR␣1⫹/⫹ or TR␣1PV/⫹ mice treated as indicated and then quantitated by real-time PCR using primers specific for (A) D1, (B) Spot14, or (C) ␣-GPD, whereas primers that amplified ␤-actin were used to normalize for the total amount of RNA in each sample. Results are the mean of two mice ⫾ the range for the hypothyroid and T3-treated groups and are the mean of five to six mice ⫾ SE for control groups. P values shown were determined using ANOVA.

experiments were performed in C57BL/6J mice. As described earlier, mice were rendered hypothyroid and then treated with T3 for 3 d. Comparable results were obtained, with the T3-dependent increase in D1 mRNA being considerably greater than that of either Spot14 or ␣-GPD. After 3 d of T3 treatment, D1 mRNA levels were 33 times greater than

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those of the hypothyroid starting point, whereas ␣-GPD and Spot14 levels both increased to only 5.5 times those of their respective hypothyroid controls (Fig. 4). TR␣ plays a role in both the positive regulation of Dio3 and the negative regulation of Dio2 by T3 in the cerebral cortex

We also wanted to study T3-dependent deiodinase regulation in TR␣1PV/⫹ mice in brain, which expresses D2 and D3 and is primarily regulated by TR␣ (12, 13). Although others have reported very low D1 activity in this tissue in mice (14), we found no D1 activity (⬍15 fmol/min/mg) in the cerebral cortex of both TR␣1⫹/⫹ and TR␣1PV/⫹ mice. Next, we studied Dio3, a gene positively regulated by T3 (15). Despite higher serum T3 levels, the TR␣1PV/⫹ mice had similar D3 activity to that of the TR␣1⫹/⫹ control group (Fig. 5A). D3 activity was not significantly decreased in both groups of hypothyroid animals. Strikingly, although D3 activity increased 5.8-fold with T3 treatment in the TR␣1⫹/⫹ mice, no significant increase was observed in the T3-treated TR␣1PV/⫹ mice. To study the regulation of the Dio2 gene in the TR␣1PV/⫹ mice, D2 mRNA and enzyme activity were measured because this gene is negatively regulated by T3 at both the transcriptional level and the T4 at the posttranslational level (1). Our results indicate that there is no difference in either D2 activity or mRNA between the control groups of the TR␣1PV/⫹ and TR␣1⫹/⫹ mice (Fig. 5B). In the hypothyroid TR␣1⫹/⫹ mice, D2 activity was increased 3.5-fold relative to untreated controls, with no change in D2 mRNA levels, which is compatible with this enzyme being primarily posttranslationally regulated by thyroid hormone (16). On the other hand, in hypothyroid TR␣1PV/⫹ animals, both D2 activity and mRNA were markedly increased relative to both control TR␣1PV/⫹ (D2 activity, 13-fold, P ⬍ 0.01; D2 mRNA, 2.4-fold) and hypothyroid TR␣1⫹/⫹ animals (D2 activity,

FIG. 5. D3 activity, D2 activity, and mRNA levels in the cerebral cortex of hypothyroid (hypo), control (ctrl), and T3-treated (T3) TR␣1⫹/⫹ and TR␣1PV/⫹ mice. TR␣1⫹/⫹ or TR␣1PV/⫹ mice were treated as indicated, and D3 enzyme activity (A) or D2 enzyme activity and mRNA levels (B) were measured. Enzyme activity shown is the mean of three to five mice per group ⫾ SE and is indicated by the gray bars. P values shown were determined using ANOVA. For determination of D2 mRNA levels, RNA was extracted from the cerebral cortex of TR␣1⫹/⫹ or TR␣1PV/⫹ mice treated as indicated and then quantitated by real-time PCR using primers specific for D2, whereas primers that amplified ␤-actin were used to normalize for the total amount of RNA in each sample. Values shown are represented by the striped bars and are the mean ⫾ SE for three mice from each group, with the exception of the TR␣1⫹/⫹ control group, where the value shown is the mean of two mice and the range is indicated. None of the differences in D2 mRNA levels were significant by ANOVA.

3-fold, P ⬍ 0.01; D2 mRNA, 4.9-fold). Keeping in mind that the T3-treated group of mice was originally at a hypothyroid starting point, in the TR␣1⫹/⫹ animals, T3 treatment caused a slight but not significant decrease in D2 mRNA and activity. In contrast to the results with the Dio3 gene, where T3 treatment was ineffective at inducing gene expression in the TR␣1PV/⫹ animals, T3 treatment did decrease D2 activity and mRNA levels by 29 and 87%, respectively, relative to that of the hypothyroid TR␣1PV/⫹ animals. Discussion FIG. 4. Hepatic D1, Spot14, and ␣-GPD mRNA levels in hypothyroid, control, and T3-treated C57BL/6J mice. C57BL/6J mice were treated with 0.1% MMI and 1% NaClO4 in their drinking water for 21 d (hypo), untreated (control), or rendered hypothyroid for a total of 24 d and then injected with 2 ␮g T3/d for 3 d on d 22–24 (T3 treated) and killed 24 h after the last T3 injection. Liver mRNA was extracted and then quantitated by real-time PCR using primers specific for D1, Spot14, or ␣-GPD, whereas primers that amplified ␤-actin were used to normalize for the total amount of RNA in each sample. Values indicated are the mean of four mice ⫾ SE for each group. Numbers above the bars indicate the fold expression relative to hypothyroid levels for the same gene.

The mild hypothalamic-pituitary resistance caused by the introduction of the dominant negative PV mutation into one allele of the TR␣ gene, as in the TR␣1PV/⫹ mouse (6), results in a mouse with 1.9-fold increase in serum T3, thus providing a unique opportunity for studying the effects of chronic mild hyperthyroidism on gene expression by the wild-type TR␤ receptor. Two critical observations were made using these animals. First, expression of hepatic D1 mRNA is markedly increased as a result of the elevated serum T3, whereas two other well-characterized hepatic T3-responsive genes, Spot14


and ␣-GPD (17–20), were increased to a much smaller extent. Second, this marked elevation of D1 is a reflection of a greater incremental response of the Dio1 gene to the elevated T3 in these animals and not due to any non-T3-dependent alterations in the level of hepatic Dio1 expression. Under hypothyroid conditions, liver D1 decreased in the TR␣1PV/⫹ animals to levels identical to those of the TR␣1⫹/⫹ animals, indicating that the observed elevation in D1 is not constitutive or due to some other T3-independent mechanism (Fig. 2A). In addition, hepatic D1 mRNA and activity are greatly increased in both T3-treated TR␣1⫹/⫹ and TR␣1PV/⫹ mice, confirming the normal T3 responsiveness of the Dio1 gene in the liver of the TR␣1PV/⫹ mice (Fig. 2A). Similar results are observed in the kidney, with D1 activity being 2.8-fold higher in the TR␣1PV/⫹ control mice than in the TR␣1⫹/⫹ animals, although this difference is not statistically significant (Fig. 2B). Furthermore, although kidney D1 activity is not significantly decreased in hypothyroidism in either mouse strain, D1 activity does increase markedly with T3 treatment. These results may reflect tissue-specific differences in D1 regulation because it has been shown in mice that a component of kidney D1 basal expression is TR independent (21). Alternatively, these results might also reflect the greater expression level of a dominant negative mutant TR␣ in this tissue blunting the effects of thyrotoxicosis or an isoform-preferential inhibition by the mutant TR␣ receptor (22–25). Our results in liver and kidney parallel those observed in another mouse model with chronically elevated serum T3 and wild-type TR␤ receptor, the TR␣⫺/⫺ mouse (21). Notably, two other mouse models with different dominant negative TR␣ mutations exhibit some similarities in phenotype to the TR␣1PV/⫹ mice, including an elevated TSH and slightly elevated T3, but D1 was not measured in either of these models (26, 27). The dominant negative effects of the mutant TR␣ PV receptor appear to be minimal on basal and T3-induced Dio1 expression in the liver, presumably due to TR␤ being the primary receptor isoform expressed in this tissue (21, 22, 28 –30). Hence, it is not surprising that a homozygous mouse model in which wild-type TR␤ has been replaced with a TR␤ receptor containing the PV mutation (TR␤PV/PV) has undetectable D1 mRNA levels despite a 9-fold elevated serum T3 (31). In fact, even when the TR␤ PV mutation is heterozygous (TR␤PV/⫹) and serum T3 is elevated to the same extent as the TR␣1PV/⫹ mice (Table 1), D1 mRNA levels are the same as in wild-type mice (31). It is notable that the expression of several other T3-responsive genes, such as Spot14, ␣-GPD (Fig. 3), and malic enzyme (6), is not elevated to the same extent as Dio1 in the liver of TR␣1PV/⫹ mice. It is possible that TR isoform-specific gene preferences could result in a selective inhibition by the TR␣ PV mutant receptor of these genes, thus mitigating the effects of the elevated serum T3 levels in the TR␣1PV/⫹ mice. However, recent microarray data indicate that the amount of the TR isoform, not the isoform type per se, is the primary determinant controlling most T3-regulated hepatic gene expression (32). Thus, one would expect the dominant negative effects of the mutant TR␣ PV receptor to be minimal in the liver. Thus, either a greater overall responsiveness or sensitivity


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of Dio1 to T3 could account for this striking difference. Although the present experiments do not directly address the issue of T3 sensitivity, our results with the TR␣1PV/⫹, TR␣1⫹/⫹, and C57BL/6J mice indicate that the overall T3 responsiveness of Dio1 is far greater than that of Spot14 and ␣-GPD. Notably, all of the differential increase in Dio1 expression occurs at T3 concentrations above euthyroid levels, whereas the fold increase in expression from hypothyroid to euthyroid is similar for all genes studied (Fig. 4). Still, it is not clear at this point whether the greater T3 responsiveness of Dio1 is specifically due to pre- or posttranscriptional differences among these genes. Although T3 does not change the half-life of D1 mRNA, nuclear run-on experiments have shown a 2- to 3-fold increase in mouse Dio1 expression after 2 h of T3 treatment (5, 33). However, the nature of the mouse Dio1 thyroid hormone response element and its role in the marked responsive of this gene to T3 are still to be defined. Recent microarray studies, which did not include Dio1 in the gene matrix (Yen, P., personal communication), have shown that Spot14 was the mouse hepatic gene with the greatest mRNA increase after 6 h of T3 treatment (34). Thus, it is notable that we found that the T3-mediated increase in Dio1 expression is at least 5 times that of Spot14. Others have also shown that Spot14 levels are increased 6-fold after of 2 h of T3 treatment (35). However, after 5 d of T3 treatment, Spot14 levels had fallen to 2-fold (35), suggesting that the magnitude of the observed response to T3 is influenced by the duration of T3 treatment. In that report (35), which used conditions of T3 treatment identical to the present investigation, the largest increase in T3-mediated gene expression relative to hypothyroid levels was 7.8-fold (Nudix 7 gene). This is still markedly less than the approximately 30- to 200-fold T3-induced increase in Dio1 expression. To determine whether the two other deiodinase genes, which are positively (Dio3) and negatively (Dio2) regulated by T3, are as sensitive to chronic T3 elevation as Dio1, we then turned to brain, which, unlike liver, predominantly expresses TR␣ (28, 36). Although treatment with T3 increased D3 activity about 4-fold in the TR␣1⫹/⫹ mice, no change was observed in the TR␣1PV/⫹ mice, illustrating the potent dominant negative effects of the TR␣ PV mutant receptor in this tissue (Fig. 5A). No difference in D3 activity was observed between the untreated TR␣1⫹/⫹ and TR␣1PV/⫹ mice, despite the difference in serum T3 values. Whether this is a reflection of most of the T3 in the brain being provided by intracellular T4 to T3 conversion by D2 (37), whether the Dio3 gene is less responsive to T3 than Dio1, or whether this is simply another manifestation of the dominant negative effects of the mutant TR␣ is unknown. D3 did not decrease in either the TR␣1PV/⫹ or TR␣1⫹/⫹ mice with hypothyroidism, as has been previously described in mice (14) but not rats (38). The negative regulation of Dio2 by T3 was impaired in the cerebral cortex of the TR␣1PV/⫹ mice, as demonstrated by the marked increase in D2 in these mice in the hypothyroid state, relative to the TR␣1⫹/⫹ animals (Fig. 5B). This is in agreement with microarray studies of T3-regulated genes using the TR␤PV/PV mouse showing that the most common type of gene dysregulation was the inappropriately elevated expression of genes negatively regulated by T3 (39). Thus, one could speculate that the limited T3 availability during hypothy-

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roidism combined with the expression of mutant TR␣ PV receptor, which is unable to bind T3, would result in higher basal levels of Dio2 expression mediated through unliganded TR␣. However, it is interesting that, with T3 treatment, one allele of the wild-type TR␣ is able to effectively mediate Dio2 suppression, whereas the T3-mediated enhancement of Dio3 expression under the same conditions was impaired (Fig. 5). This illustrates how TR-mediated relief of repression and transactivation are not simply opposite images of the same paradigm. A striking observation was the much higher serum T3 levels (8.5-fold and 5.3-fold in two separate experiments) found in the T3-treated TR␣1PV/⫹ mice relative to normal littermates 24 h after T3 treatment. One explanation that can be ruled out is higher levels of T3-binding plasma proteins because the free T3 fraction is 28% higher in the TR␣1PV/⫹ mice than in TR␣1⫹/⫹ mice. An alternative explanation is that T3 clearance is significantly reduced in the TR␣1PV/⫹ mice relative to that of the TR␣1⫹/⫹ controls. The major deiodinative pathways for T3 clearance are via inner-ring deiodination by D3 and D1 (1). However, the T3-mediated induction of D1 in the TR␣1PV/⫹ mice is normal, thus pointing to a significant role of D3 in T3 clearance. Thus, T3dependent induction of D3 found in tissues, such as the skin and the central nervous system, could also be playing a significant role in T3 metabolism (1). Consistent with a role of D3 in T3 clearance, patients with D3-overexpressing hepatic hemangiomas display a marked acceleration of T3 clearance (40). The main reason for serum T3 to be increased in the TR␣1PV/⫹ mice is a central resistance to thyroid hormone, resulting in increased serum TSH and thyroid hormone production (6). Still, we cannot exclude the possibility that the failure of D3 to increase in response to the elevated serum T3 is not a compounding factor in the 1.9-fold increase in serum T3 in these animals (Fig. 5A, Table 1). Additionally, D1 contributes about 30% of the daily T3 production in the mouse (1), thus the enhanced D1 activity found in the TR␣1PV/⫹ mice could also contribute to the increased serum T3 levels found in these animals. However, when the TR␣1PV/⫹ mice were made hypothyroid, D1 activity was not different between TR␣1PV/⫹ and TR␣1⫹/⫹mice, indicating that the elevation of D1 in the TR␣1PV/⫹ is the result, and not the cause, of the elevated serum T3 levels found in these animals (Fig. 2A). In conclusion, the present studies, using this unique model of chronic hyperthyroidism, have allowed us to uncover novel information about the mechanism of T3 action that might otherwise not have been discernable. These studies suggest that the T3 regulation of the Dio2 and Dio3 genes in brain is mediated through TR␣ and further indicate that the T3-mediated induction of Dio3 expression may also play a previously unrecognized role in T3 clearance. Notably, these studies indicate that the increased hepatic D1 levels found in the TR␣1PV/⫹ mouse are a result of the mild elevation in serum T3 levels and, further, that the Dio1 gene is substantially more responsive to T3 than any other mouse gene identified to date. Although the mechanism for this response remains to be elucidated, our results suggest that, in the


mouse, Dio1 expression may have a use similar to that of TSH in assessing the thyroid status of the peripheral tissues. Acknowledgments We thank Stephen A. Huang, M.D., and Michelle Mulcahey for assistance with the D3 enzyme assay. Received October 21, 2004. Accepted December 1, 2004. Address all correspondence and requests for reprints to: Ann Marie Zavacki, Ph.D., Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM 641, Boston, Massachusetts 02115. E-mail: azavacki@rics. bwh.harvard.edu. This work was supported by Grants DK44128 (to P.R.L.) and DK65765 (to A.M.Z.) from National Institute of Diabetes and Digestive and Kidney Diseases.

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