Thyroxine-induced expression of pyroglutamyl peptidase II and inhibition of TSH release precedes suppression of TRH mRNA and requires type 2 deiodinase.

Suppression of TSH release from the hypothyroid thyrotrophs is one of the most rapid effects of 3,3',5'-triiodothyronine (T(3)) or thyroxine (T(4)). It is initiated within an hour, precedes the decrease in TSHß mRNA inhibition and is blocked by inhibitors of mRNA or protein synthesis. TSH elevation in primary hypothyroidism requires both the loss of feedback inhibition by thyroid hormone in the thyrotrophs and the positive effects of TRH. Another event in this feedback regulation may be the thyroid hormone-mediated induction of the TRH-inactivating pyroglutamyl peptidase II (PPII) in the hypothalamic tanycytes. This study compared the chronology of the acute effects of T(3) or T(4) on TSH suppression, TRH mRNA in the hypothalamic paraventricular nucleus (PVN), and the induction of tanycyte PPII. In wild-type mice, T(3) or T(4) caused a 50% decrease in serum TSH in hypothyroid mice by 5  h. There was no change in TRH mRNA in PVN over this interval, but there was a significant increase in PPII mRNA in the tanycytes. In mice with genetic inactivation of the type 2 iodothyronine deiodinase, T(3) decreased serum TSH and increased PPII mRNA levels, while T(4)-treatment was ineffective. We conclude that the rapid suppression of TSH in the hypothyroid mouse by T(3) occurs prior to a decrease in TRH mRNA though TRH inactivation may be occurring in the median eminence through the rapid induction of tanycyte PPII. The effect of T(4), but not T(3), requires the type 2 iodothyronine deiodinase.

Type-2 iodothyronine 5'deiodinase in skeletal muscle of C57BL/6 mice. I. Identity, subcellular localization, and characterization.

RT-PCR shows that mouse skeletal muscle contains type-2 iodothyronine deiodinase (D2) mRNA. However, the D2 activity has been hard to measure. Except for newborn mice, muscle homogenates have no detectable activity. However, we have reported D2 activity in mouse muscle microsomes. As the mRNA, activity is higher in slow- than in fast-twitch muscle. We addressed here the major problems in measuring D2 activity in muscle by: homogenizing muscle in high salt to improve yield of membranous structures; separating postmitochondrial supernatant between 38 and 50% sucrose, to eliminate lighter membranes lacking D2; washing these with 0.1 M Na(2)CO(3) to eliminate additional contaminating proteins; pretreating all buffers with Chelex, to eliminate catalytic metals; and eliminating the EDTA from the assay, as this can bind iron that enhances dithiothreitol oxidation and promotes peroxidation reactions. Maximum velocity of T(3) generation by postgradient microsomes from red muscles was approximately 1100 fmol/(h · mg) protein with a Michaelis-Menten constant for T(4) of 1.5 nM. D2-specific activity of Na(2)CO(3)-washed microsomes was 6-10 times higher. The enrichment in D2 activity increased in parallel with the capacity of microsomes to load (sarco/endoplasmic reticulum Ca(2+)-ATPase) and bind Ca(2+) (calsequestrin), indicating that D2 resides in the inner sarcoplasmic reticulum, close to the nuclei. The presence of D3 in the sarcolemma suggests that the most of D2-generated T(3) acts locally. Estimates from maximum velocity, Michaelis-Menten constant, and muscle T(4) content suggest that mouse red, type-1, aerobic mouse muscle fibers can generate physiologically relevant amounts of T(3) and, further, that muscle D2 plays an important role in thyroid hormone-dependent muscle thermogenesis.

Type-2 iodothyronine 5'deiodinase (D2) in skeletal muscle of C57Bl/6 mice. II. Evidence for a role of D2 in the hypermetabolism of thyroid hormone receptor alpha-deficient mice.

Mice with ablation of the Thra gene have cold intolerance due to an as yet undefined defect in the activation of brown adipose tissue (BAT) uncoupling protein (UCP). They develop an alternate form of facultative thermogenesis, activated at temperatures below thermoneutrality and associated with hypermetabolism and reduced sensitivity to diet-induced obesity. A consistent finding in Thra-0/0 mice is increased type-2 iodothyronine deiodinase (D2) mRNA in skeletal muscle and other tissues. With an improved assay to measure D2 activity, we show here that this enzyme activity is increased in proportion to the mRNA and as a function of the ambient cold. The activation is mediated by the sympathetic nervous system in Thra-0/0, as it is in wild-type genotype mice, but the sympathetic nervous system effect is greater in Thra-0/0 mice. Using D2-ablated mice (Dio2-/-), we reported elsewhere and show here that, in spite of sharing a severe deficiency in BAT thermogenesis with Thra-0/0 and UCP1-knockout mice, they do not have an increase in oxygen consumption, and they gain more weight than wild-type controls when fed a high-fat diet. UCP3 mRNA is highly responsive to thyroid hormone, and it is increased in Thra-0/0 mice, particularly when fed high-fat diets. We show here that muscle UCP3 mRNA in hypothyroid Thra-0/0 mice is responsive to small dose-short regimens of T(4), indicating a role for locally, D2-generated T(3). Lastly, we show that bile acids stimulate not only BAT but also muscle D2 activity, and this is associated with stimulation of muscle UCP3 mRNA expression provided T(4) is present. These observations strongly support the concept that enhanced D2 activity in Thra-0/0 plays a critical role in their alternate form of facultative thermogenesis, stimulating increased fat oxidation by increasing local T(3) generation in skeletal muscle.

Physiological role and regulation of iodothyronine deiodinases: a 2011 update.

T4 is a prohormone secreted by the thyroid. T4 has a long half life in circulation and it is tightly regulated to remain constant in a variety of circumstances. However, the availability of iodothyronine selenodeiodinases allow both the initiation or the cessation of thyroid hormone action and can result in surprisingly acute changes in the intracellular concentration of the active hormone T3, in a tissue- specific and chronologically-determined fashion, in spite of the constant circulating levels of the prohormone. This fine-tuning of thyroid hormone signaling is becoming widely appreciated in the context of situations where the rapid modifications in intracellular T3 concentrations are necessary for developmental changes or tissue repair. Given the increasing availability of genetic models of deiodinase deficiency, new insights into the role of these important enzymes are being recognized. In this review, we have incorporated new information regarding the special role played by these enzymes into our current knowledge of thyroid physiology, emphasizing the clinical significance of these new insights.

Overexpression of type 2 iodothyronine deiodinase in follicular carcinoma as a cause of low circulating free thyroxine levels.

Thyroid function is normally undisturbed in patients with thyroid carcinoma. We have identified three patients with large or widely metastatic follicular thyroid carcinoma who had a persistently increased ratio of serum T(3) to T(4) in the absence of autonomous production of T(3) by the tumor. To investigate the possibility of tumor-mediated T(4) to T(3) conversion, we assayed types 1 and 2 iodothyronine selenodeiodinase (D1 and D2) activity in a 965-g follicular thyroid carcinoma resected from one of these patients. The V(max) for D2 was 8-fold higher than in normal human thyroid tissue. Resection of this tumor, leaving the left thyroid lobe intact, normalized the serum T(3) to T(4) ratio. In two other patients, treatment with sufficient levothyroxine to suppress TSH was associated with a high normal T(3) and a subnormal free T(4) index. In one, concomitant administration of the D1 inhibitors, propylthiouracil and propranolol, did not decrease the elevated serum T(3) to T(4) ratio. These data illustrate that increased T(4) to T(3) conversion in follicular thyroid carcinomas, probably by D2, can cause a significant perturbation in peripheral thyroid hormone concentrations.

The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue.

Type 2 iodothyronine deiodinase (D2) is a selenoenzyme, the product of the recently cloned cAMP-dependent Dio2 gene, which increases 10- to 50-fold during cold stress only in brown adipose tissue (BAT). Here we report that despite a normal plasma 3,5,3'-triiodothyronine (T3) concentration, cold-exposed mice with targeted disruption of the Dio2 gene (Dio2(-/-)) become hypothermic due to impaired BAT thermogenesis and survive by compensatory shivering with consequent acute weight loss. This occurs despite normal basal mitochondrial uncoupling protein 1 (UCP1) concentration. In Dio2(-/-) brown adipocytes, the acute norepinephrine-, CL316,243-, or forskolin-induced increases in lipolysis, UCP1 mRNA, and O(2) consumption are all reduced due to impaired cAMP generation. These hypothyroid-like abnormalities are completely reversed by a single injection of T3 14 hours earlier. Recent studies suggest that UCP1 is primarily dependent on thyroid hormone receptor beta (TR beta) while the normal sympathetic response of brown adipocytes requires TR alpha. Intracellularly generated T3 may be required to saturate the TR alpha, which has an approximately fourfold lower T3-binding affinity than does TR beta. Thus, D2 is an essential component in the thyroid-sympathetic synergism required for thermal homeostasis in small mammals.

Transcriptional regulation of iodothyronine deiodinases during embryonic development.

A single dose of chicken growth hormone (cGH) or dexamethasone acutely increases circulating T(3) levels in 18-day-old chicken embryos through a reduction of hepatic type III iodothyronine deiodinase (D3). The data in the present study suggest that this decrease in D3 is induced by a direct downregulation of hepatic D3 gene transcription. The lack of effect of cGH or dexamethasone on brain and kidney D3 activity, furthermore suggests that both hormones affect peripheral thyroid hormone metabolism in a tissue specific manner. Dexamethasone administration also results in an increase in brain type II iodothyronine deiodinase (D2) activity and mRNA levels that is also regulated at a transcriptional level. In contrast, however, cGH has no effect on brain D2 activity, thereby suggesting that either GH cannot pass through the blood-brain barrier in chicken or that cGH and dexamethasone regulate thyroid hormone deiodination by different mechanisms. In addition, the very short half-life of D2 and D3 (t(1/2)<1 h) in comparison with the longer half life of type I iodothyronine deiodinase (D1, t(1/2)>8 h), allows for D2 and D3 to play a more prominent role in the acute regulation of peripheral thyroid hormone metabolism than D1.

The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line.

Types 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with (75)Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have approximately 40-fold higher levels of hDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T(4)) to 3,5,3'-triiodothyronine with typical characteristics of D2 such as low K(m) (T(4)), 1.3 nm, resistance to propylthiouracil, and a short half-life ( approximately 30 min). D2 activity is approximately 30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from the Dio2 mRNA sequence precipitates a (75)Se protein of the predicted 31-kDa size from (75)Se-labeled mesothelioma cells. Bromoadenosine 3'5' cyclic monophosphate increases D2 activity and (75)Se-p31 approximately 2.5-fold whereas substrate (T(4)) reduces both D2 activity and (75)Se-p31 approximately 2-3-fold. MG132 or lactacystin (10 microm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and (75)Se-p31 3-4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T(4)) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T(4) activation.

Regional physiological adaptation of the central nervous system deiodinases to iodine deficiency.

The goal of the present investigation was to analyze the types 2 (D2) and 3 (D3) iodothyronine deiodinases in various structures within the central nervous system (CNS) in response to iodine deficiency. After 5-6 wk of low-iodine diet (LID) or LID + 2 microg potassium iodide/ml (LID + KI; control), rats' brains were processed for in situ hybridization histochemistry for D2 and D3 mRNA or dissected, frozen in liquid nitrogen, and processed for D2 and D3 activities. LID did not affect weight gain or serum triiodothyronine, but plasma thyroxine (T4) was undetectable. In the LID + KI animals, D3 activities were highest in the cerebral cortex (CO) and hippocampus (HI), followed by the olfactory bulb and was lowest in cerebellum (CE). Iodine deficiency decreased D3 mRNA expression in all CNS regions, and these changes were accompanied by three- to eightfold decreases in D3 activity. In control animals, D2 activity in the medial basal hypothalamus (MBH) was similar to that in pituitary gland. Of the CNS D2-expressing regions analyzed, the two most responsive to iodine deficiency were the CO and HI, in which an approximately 20-fold increase in D2 activity occurred. Other regions, i.e., CE, lateral hypothalamus, MBH, and pituitary gland, showed smaller increases. The distribution of and changes in D2 mRNA were similar to those of D2 activity. Our results indicate that decreases in the expression of D3 and increases in D2 are an integral peripheral component of the physiological response of the CNS to iodine deficiency.

Relation of severity of maternal hypothyroidism to cognitive development of offspring.

BACKGROUND: An association between maternal subclinical hypothyroidism and low intelligence quotient (IQ) in the offspring has recently been shown. OBJECTIVE: To provide evidence for the causality of the association by testing the hypothesis that severity of maternal hypothyroidism correlates inversely with IQ of the offspring. METHODS: IQ scores were compared among 8 year old offspring of 124 control mothers whose thyroid stimulating hormone (TSH) concentrations were < 98th percentile of a cohort of 25,000 mothers at 17 weeks gestation, of 28 untreated hypothyroid women whose TSH was between the 98th and 99.85th percentiles, and of 20 untreated women whose TSH concentration was > or = 99.85th percentile. RESULTS: Mean (SD) IQs for each group of children (in ascending order of maternal TSH concentration) were 107 (13), 102 (15), and 97 (14). The difference between the extremes was significant (p = 0.003). The percentage of children with IQs > 1 SD below the control mean was 15, 21, and 50 respectively (p = 0.003). The odds ratio of having an IQ > 1 SD below the control mean, after controlling for socioeconomic status, was 4.7 (p = 0.006) for the third group compared with the controls. CONCLUSIONS: The inverse correlation between severity of maternal hypothyroidism and IQ of the offspring supports a causal relation and makes the need to screen for and treat pregnant women for hypothyroidism even more compelling.

The human, but not rat, dio2 gene is stimulated by thyroid transcription factor-1 (TTF-1).

Types 1 and 2 iodothyronine deiodinases (D1 and D2) catalyze the production of T(3) from T(4). D2 mRNA is abundant in the human thyroid but very low in adult rat thyroid, whereas D1 activity is high in both. To understand the molecular regulation of these genes in thyroid cells, the effect of thyroid transcription factor 1 (TTF-1) and the paired domain-containing protein 8 (Pax-8) on the transcriptional activity of the deiodinase promoters were studied. Both the approximately 6.5-kb hdio2 sequence and its most 3' 633 bp were activated 10-fold by transiently expressed TTF-1 in COS-7 cells, but the hdio1 was unaffected. Surprisingly, the response of the rdio2 gene to TTF-1 was only 3-fold despite the 73% identity with the proximal 633-bp region of hdio2 including complete conservation of a functional cAMP response element at -90. Neither human nor rat dio2 nor human dio1 was induced by Pax-8. The binding affinity of four putative TTF-1 binding sites in hdio2 were compared by a semiquantitative gel retardation assay using in vitro expressed TTF-1 homeodomain protein. Only two sites, D and C1 (both of which are absent in rdio2), had significant affinity. Functional analyses showed that both sites are required for the full response to TTF-1. These results can explain the differential expression of dio2 in thyroid and potentially other tissues in humans and rats.

Type 2 iodothyronin deiodinase transgene expression in the mouse heart causes cardiac-specific thyrotoxicosis.

Type 2 iodothyronine deiodinase (D(2)) catalyzes intracellular 3, 5, 3' triiodothyronine (T(3)) production from thyroxine (T(4)), and its messenger RNA mRNA is highly expressed in human, but not rodent, myocardium. The goal of this study was to identify the effects of D(2) expression in the mouse myocardium on cardiac function and gene expression. We prepared transgenic (TG) mice in which human D(2) expression was driven by the alpha-MHC promoter. Despite high myocardial D(2) activity, myocardial T(3) was, at most, minimally increased in TG myocardium. Although, plasma T(3) and T(4), growth rate as well as the heart weight was not affected by TG expression, there was a significant increase in heart rate of the isolated perfused hearts, from 284 +/-12 to 350 +/- 7 beats/min. This was accompanied by an increase in pacemaker channel (HCN2) but not alpha-MHC or SERCA II messenger RNA levels. Biochemical studies and (31)P-NMR spectroscopy showed significantly lower levels of phosphocreatine and creatine in TG hearts. These results suggest that even mild chronic myocardial thyrotoxicosis, such as may occur in human hyperthyroidism, can cause tachycardia and associated changes in high energy phosphate compounds independent of an increase in SERCA II and alpha-MHC.

The role of selenocysteine 133 in catalysis by the human type 2 iodothyronine deiodinase.

Human type 2 iodothyronine deiodinase (hD2) catalyzes the activation of T4 to T3. D2, like types 1 and 3 deiodinases, contains selenocysteine (Sec) in the highly conserved active center at position 133. To evaluate the contribution of Sec133 to the catalytic properties of hD2, we generated mutants in which cysteine (Cys) or alanine (Ala) replaced Sec133. The Km (T4) of Cys133D2 was 2.1 microM, strikingly higher than that of native D2 (1.4 nM). In contrast, the relative turnover number was 10-fold lower for Cys133D2, illustrating the greater potency of Se than S in supporting catalysis. The AlaD2 mutant was inactive. Studies in intact cells transiently expressing the native or Cys133D2 enzyme exhibited saturation kinetics expected from the Km as measured under in vitro conditions, indicating rapid equilibration of extracellular and intracellular T4. Blockade of the NTCP, OATP1-3, and LST-1 transporters with 10 mM sodium taurocholate did not alter the deiodination rate of T4 by Cys133D2 transiently expressed in intact cells, suggesting that intracellular transport of T4 is not rate limiting. These results illustrate that selenium plays a critical role in deiodination catalyzed by hD2.

Usefulness of ultrasonography in the management of nodular thyroid disease.

BACKGROUND: Fine-needle aspiration biopsy is the standard diagnostic test for evaluating possible malignancy in a thyroid nodule. OBJECTIVE: To evaluate the role of routine ultrasonography in the management of nodular thyroid disease. DESIGN: Retrospective chart review. SETTING: Multidisciplinary thyroid nodule clinic (endocrinology and radiology). PATIENTS: Patients with suspected nodular thyroid disease or suspected recurrent thyroid cancer referred between October 1995 and March 1997. All patients had thyroid ultrasonography and ultrasonography-guided fine-needle aspiration biopsy of nodules at least 1 cm in maximum diameter. MEASUREMENTS: Medical records, ultrasonography findings, cytology reports, and histologic reports were reviewed. Ultrasonography findings were compared with the referring physician's findings on physical examination. RESULTS: 223 patients were seen in the clinic. A total of 209 fine-needle aspiration biopsies were performed on 156 patients. Among 50 of 114 patients referred for a solitary nodule, ultrasonography detected additional nonpalpable nodules at least 1 cm in diameter in 27 and determined that no nodules required aspiration in 23. Of 59 patients referred for a diffuse goiter or a multinodular gland, ultrasonography detected discrete nodules at least 1 cm in diameter that required aspiration in 39 and determined that aspiration was unnecessary in 20. CONCLUSIONS: Ultrasonography altered the clinical management for 63% of the patients (109 of 173) referred to the thyroid nodule clinic after abnormal results on thyroid physical examination.

Selective proteolysis of human type 2 deiodinase: a novel ubiquitin-proteasomal mediated mechanism for regulation of hormone activation.

We investigated the mechanism by which T4 regulates its activation to T3 by the type 2 iodothyronine deiodinase (D2). D2 is a short- lived (t1/2 50 min), 31-kDa endoplasmic reticulum (ER) integral membrane selenoenzyme that generates intracellular T3. Inhibition of the ubiquitin (Ub) activating enzyme, E1, or MG132, a proteasome blocker, inhibits both the basal and substrate-induced acceleration of D2 degradation. Using a catalytically active transiently expressed FLAG-tagged-NH2-D2, we found rapid synthesis of high molecular mass (100-300 kDa) Ub-D2 conjugates that are catalytically inactive. Ub-D2 increases when cells are exposed to D2 substrate or MG132 and disappears rapidly after E1 inactivation. Fusion of FLAG epitope to the COOH terminus of D2 prolongs its half-life approximately 2.5-fold and increases the levels of active and, especially, Ub-D2. This indicates that COOH-terminal modification interferes with proteasomal uptake of Ub-D2 that can then be deubiquitinated. Interestingly, the type 1 deiodinase, a related selenoenzyme that also converts T4 to T3 but with a half-life of >12 h, is inactivated but not ubiquitinated or degraded after exposure to substrate. Thus, ubiquitination of the ER-resident enzyme D2 constitutes a specific posttranslational mechanism for T4 regulation of its own activation in the central nervous system and pituitary tissues in which D2-catalyzed T4 to T3 conversion is the major source of intracellular T3.

Distinct subcellular localization of transiently expressed types 1 and 2 iodothyronine deiodinases as determined by immunofluorescence confocal microscopy.

We compared the subcellular localization of FLAG-epitope tagged Types 1 and 2 deiodinases (D1 and D2) transiently expressed in human embryonic kidney (HEK-293) and mouse neuroblastoma (NB2A) cells. D2 is an integral membrane protein based on resistance to extraction at pH 11 with the NH2 terminus in the endoplasmic reticulum (ER). Immunofluorescence confocal microscopy using anti-FLAG and anti-GRP78/BiP antibodies showed the FLAG-D1 signal was found in the periphery of the cells and not co-localized with the ER specific marker GRP78/BiP. On the other hand, FLAG-D2 protein was found in the ER co-localized with the GRP78/BiP protein. These differential distribution patterns indicate subcellular sorting of D1 and D2 is determined by intrinsic protein sequence and can explain the ready access of D2-generated T3 to the nucleus.

DARPP-32 and CREB are present in type 2 iodothyronine deiodinase-producing tanycytes: implications for the regulation of type 2 deiodinase activity.

Type 2 iodothyronine deiodinase, an enzyme involved in the conversion of thyroxin to the biologically active 3,5, 3'-triiodothyronine, is highly concentrated in a group of specialized ependymal cells, tanycytes, lining the wall and floor of the third ventricle. As this distribution is highly reminiscent of the distribution of cells containing the phosphatase inhibitor, DARPP-32, we raised the possibility that these two proteins may coexist in tanycytes and that DARPP-32 may modulate type 2 deiodinase activity by regulating the phosphorylation state of the cAMP regulatory factor, CREB. To address this question, double-labeling histochemical studies were performed for type 2 deiodinase mRNA and DARPP-32 immunoreactivity (IR), or DARPP-32- and CREB-IR in the same tissue sections. Type 2 deiodinase mRNA was found in the cell bodies of all DARPP-32-immunolabeled tanycytes. Both type 2 deiodinase mRNA and DARPP-32-IR also extended into tanycyte processes that ramified in the arcuate nucleus and median eminence, in close association with blood vessels and portal capillaries. In contrast, type 2 deiodinase mRNA was not present in the same cells that contained DARPP-32-IR in the pituitary gland. All tanycytes containing DARPP-32-IR also contained CREB-IR in their nucleus. Since type 2 deiodinase activity can be induced by substances that increase cAMP, we hypothesize that DARPP-32 may regulate the activity of type 2 deiodinase by prolonging the activation of CREB. Selectivity for the colocalization of these factors to tanycytes but not the pituitary gland, may explain the heterogeneous response of type 2 deiodinase activity in these two loci in response to specific stimuli such as fasting.

Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme's active center.

Type 2 iodothyronine deiodinase (D2) catalyzes the first step in thyroid hormone action, the deiodination of T4 to T3. Endogenous D2 activity is posttranslationally regulated by substrate that accelerates its degradation through the ubiquitin-proteasome pathway. To understand how D2 activity correlates with D2 protein during its normal decay and rT3-induced down-regulation, HEK-293 cells, transiently expressing human D2, were labeled with Na75SeO3 and then treated with 100 microM cycloheximide (CX), 30 nM rT3, and/or 10 microM MG132, a specific proteasome inhibitor, for 2-4 h. D2 protein and enzyme activity changed in parallel, disappearing with a half-life of 2 h in the presence of CX, or 1 h when CX + rT3 were combined. Treatment with MG132 blocked these effects. We created selenocysteine (Sec) 133 to cysteine (Cys) or alanine (Ala) D2 mutants, without changing Sec 266. The CysD2 activity and protein levels were also parallel, with a similar half-life of approximately 2 h, whereas the rT3-induced D2 down-regulation required approximately 1000-fold higher rT3 concentration (30 microM) due to a proportionally higher Michaelis constant of CysD2. In similar experiments, the AlaD2 mutant retained the short half-life but was not catalytically active and not susceptible to rT3-accelerated degradation. We conclude that substrate-induced loss of D2 activity is due to proteasomal degradation of the enzyme and requires interaction with the catalytic center of the protein.

Characterization of the 5'-flanking and 5'-untranslated regions of the cyclic adenosine 3',5'-monophosphate-responsive human type 2 iodothyronine deiodinase gene.

The type 2 iodothyronine deiodinase (D2) catalyzes T4 activation. In humans, unlike rodents, it is widely expressed, and its action probably contributes to both intracellular and plasma T3 pools. We have isolated the 6.5-kb 5'-flanking region (FR) and the previously uncloned 553 nucleotides (nt) of the 5'-untranslated region (UTR) of hdio2. The 5'-UTR is complex, with three transcription start sites (TSS) (708, 31, and approximately 24 nt 5' to the ATG), an alternatively spliced approximately 300-nt intron in the 5'-UTR, and three short open reading frames 5' to the initiator ATG. The previously reported approximately 7.5-kb D2 messenger RNA (mRNA) is actually an approximately 7-kb doublet that is present in thyroid, pituitary, cardiac and skeletal muscle, and possibly brain, but with only the longer transcript in placenta. A canonical cAMP response element-binding protein-binding site is present at about 90 bp 5' to the most 5'-TSS. It accounts for the robust response of the 6.8-kb hdio2 5'-FR to protein kinase A. Forskolin increases D2 mRNA in human thyroid cells, which may explain the high D2 mRNA in Graves' thyroid and thyroid adenomas. The hdio2 gene structure and Northern blot results suggest that D2 expression is tightly controlled and tissue specific.