Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4.

The type 2 deiodinase (D2), a selenoenzyme that catalyzes the conversion of T4 to T3 via 5'-deiodination, is expressed in the pituitary, brain, brown adipose tissue (BAT), and the reproductive tract. To examine the physiological role of this enzyme, a mouse strain lacking D2 activity was developed using homologous recombination. The targeting vector contained the Neo gene in place of a 2.6-kb segment of the Dio2 gene. This segment comprises 72% of the coding region and includes the TGA codon that codes for the selenocysteine located at the active site of the enzyme. Mice homologous for the targeted deletion [D2 knockout (D2KO)] had no gross phenotypic abnormalities, and development and reproductive function appeared normal, except for mild growth retardation (9%) in males. No D2 activity was observed in any tissue in D2KO mice under basal conditions, or under those that normally induce this enzyme such as cold-exposure (BAT) or hypothyroidism (brain, BAT, and pituitary gland). Furthermore, no D2 activity was present in cultured astrocytes, nor could it be induced by treatment of the cells with forskolin. Although D2 mRNA transcripts were detected in BAT RNA obtained from cold-exposed wild-type (WT) mice, none was detected in BAT RNA from comparably-treated D2KO mice. Levels of D1 in the liver, thyroid, and pituitary were the same in WT and D2KO animals, whereas D3 activity in D2KO cerebrum was twice that in WT cerebrum. Serum T3 levels were comparable in adult WT and D2KO mice. However, serum T4 and TSH levels were both elevated significantly (40% and 100%, respectively) in the D2KO mice, suggesting that the pituitary gland of the D2KO mouse is resistant to the feedback effect of plasma T4. This view was substantiated by the finding that serum TSH levels in hypothyroid WT mice were suppressed by administration of either T4 or T3, but only T3 was effective in the D2KO mouse. The data also suggest that the clearance of T4 from plasma was reduced in the D2KO mouse. In summary, targeted inactivation of the selenodeiodinase Dio2 gene results in the complete loss of D2 activity in all tissues examined. The increased serum levels of T4 and TSH observed in D2KO animals demonstrate that the D2 is of critical importance in the feedback regulation of TSH secretion.

The type 2 iodothyronine deiodinase is expressed in the rat uterus and induced during pregnancy.

Thyroid hormones are of considerable importance for vertebrate reproductive function and during development. To further assess the role of these compounds in this capacity, we examined the expression pattern of the type 2 iodothyronine deiodinase (D2), which converts T(4) to the more active hormone T(3), in the rat uterus in both the nonpregnant and the pregnant state. D2 activity was identified as the predominant, if not only, 5'-deiodinase in the nonpregnant rat uterus. The expression of D2 messenger RNA was located by in situ hybridization to the endometrial stromal cells, where the signal was particularly enriched in the region adjacent to the epithelial cells of the uterine lumen. During pregnancy, D2 activity increased, peaking on day 17 of gestation (embryonic day 17). At that time, uterine D2 activity exceeded that in the placenta, as well as that in the fetal tissues. In the earlier stages of pregnancy before placental formation (e.g. embryonic days 10-11), D2 messenger RNA in the rat uterus was located outside the decidual tissue, which was observed, as in previous studies, to highly express the inactivating type 3 deiodinase. In summary, the rat uterus, particularly during pregnancy, seems to be a site of active thyroid hormone metabolism, presumably designed to maintain the optimal thyroid hormone environment for both the fetus and the maternal uterine tissue.

Iodothyronine sulfotransferase activity in rat uterus during gestation.

In developing mammals, we and others demonstrated that sulfation is an important pathway in the metabolism of thyroid hormone, and there is significant fetal-maternal transfer of sulfated iodothyronine. In the present study, we characterized a novel iodothyronine sulfotransferase (IST) in pregnant rat uterus. (125)I-labeled 3,3'-diiodothyronine (T(2)), T(3), rT(3), and T(4) were used as substrates with unlabeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfate donor. Sulfated iodothyronine products were separated by Sephadex LH-20 column and further identified on reverse phase HPLC. We measured IST activity in pregnant rat uterus by incubating 1 microM substrate, 50 microM PAPS, and 50 microg cytosol protein, pH 7.2, 30 min at 37 degrees C. The results show that the substrate preference of the uterine IST activity is: T(2 )> rT(3 )> T(3)> T(4); the pH optimum is 6.0 for T(2). The K(m) and V:(max) (for gestational day 21 uterus) for T(2) are 0.62 microM and 3466 pmol/mg protein/h, respectively; for PAPS the values are 2.6 microM and 1523 pmol/mg protein/h, respectively. During pregnancy, the total activities exhibit a U-shaped curve with minimum activity at day 13 of gestation; while a thermostable activity increases significantly near term. In summary, there is significant uterine IST that varies during pregnancy. The role of this uterine sulfotransferase activities in regulating the bioavailability of thyroid hormone in the developing fetus remains to be elucidated.

Effects of selenium deficiency on tissue selenium content, deiodinase activity, and thyroid hormone economy in the rat during development.

The iodothyronine deiodinases, D1, D2, and D3, all contain selenium (Se) in the form of selenocysteine at their active sites, and they play crucial roles in determining the circulating and intracellular levels of the active thyroid hormone (TH), T3. However, not only are serum T3 levels normal in Se-deficient rats but phenotypic and reproductive abnormalities are minimal, and it has been suggested that regulatory mechanisms exist to conserve Se in critical tissues. The present study was designed to determine, in rats: 1) whether the effects of Se-deficiency are greater in the fetus and neonate than in the adult; 2) whether there are tissues other than brain and thyroid in which deiodinase activities are maintained; 3) whether the maintenance of deiodinase activity in a specific tissue is associated with a concomitant preservation of Se level in that tissue; and 4) whether TH economy and general health is maintained over several generations. The tissues studied included liver, cerebrum, thyroid, pituitary, skin, brown adipose tissue, uterus, ovary, testis, placenta, and the implantation site (uterus plus contents) at E9. The results have revealed that, with the exception of liver, skin, and nonpregnant uterus, all of the tissues studied maintained substantial deiodinase activity (>50%) during prolonged Se-deficiency. Second, although the ability of a tissue to maintain deiodinase activity in the face of dietary Se deprivation was associated in some tissues with a concomitant local preservation of Se concentration, this was not the case for all tissues. Only when Se levels were decreased by more than 80% was deiodinase activity markedly decreased. Third, the effects of Se-deficiency were no greater in the fetus than in the adult; and fourth, at the level of Se-deficiency employed in this study, TH economy and general health were successfully maintained over six generations of Se-deficient rats. How Se levels are maintained in specific tissues, whether Se is sequestered in specific cells of a tissue or organ during dietary Se deprivation, and the precise mechanisms by which plasma T3 levels are maintained in Se-deficient animals remain unanswered. Further insights may be gained by using diets that are even lower in Se than those that were used herein and/or by conducting studies using radioactive forms of Se and thyroid hormones.

Regulation of type 3 iodothyronine deiodinase expression in cultured rat astrocytes: role of the Erk cascade.

The type 3 iodothyronine deiodinase (D3) metabolizes thyroid hormones to inactive metabolites in many tissues, including the brain. In the present studies, we have examined the mechanisms by which T3 (T3), retinoic acid, 12-O-tetradecanoyl phorbol 13-acetate (TPA), and basic fibroblast growth factor (bFGF) induce D3 expression in primary cultures of neonatal rat astrocytes. In untreated cells, D3 messenger RNA (mRNA) was essentially undetectable by Northern analysis and RT-PCR. However, all four agents induced expression of a 2.4-kb D3 transcript as well as D3 activity. Induction of D3 by TPA and bFGF was more rapid than that by T3 and retinoic acid, and T3 potentiated the stimulatory effects of TPA and bFGF. D3 induction by TPA was blocked by GF 109203X, an inhibitor of protein kinase C. In addition, the effects of TPA and bFGF were partially prevented by PD 98059, a specific inhibitor of MEK and the Erk signaling cascade. These studies demonstrate that multiple growth factors and hormones regulate D3 activity in cultured astrocytes by inducing D3 mRNA expression. In addition, the stimulatory effects of TPA and bFGF on D3 mRNA and activity appear to be mediated at least in part by activation of the MEK/Erk signaling cascade.

Pregnant rat uterus expresses high levels of the type 3 iodothyronine deiodinase.

Although thyroid hormones are critically important for the coordination of morphogenic processes in the fetus and neonate, premature exposure of the embryo to levels of the hormones present in the adult is detrimental and can result in growth retardation, malformations, and even death. We report here that the pregnant rat uterus expresses extremely high levels of the type 3 iodothyronine deiodinase (D3), which inactivates thyroxine and 3,3', 5-triiodothyronine by 5-deiodination. Both D3 mRNA and activity were present at the implantation site as early as gestational day 9 (E9), when expression was localized using in situ hybridization to uterine mesometrial and antimesometrial decidual tissue. At later stages of gestation, uterine D3 activity remained very high, and the levels exceeded those observed in the placenta and in fetal tissues. After days E12 and E13, as decidual tissues regressed, D3 expression became localized to the epithelial cells lining the recanalized uterine lumen that surrounds the fetal cavity. These findings strongly suggest that the pregnant uterus, in addition to the placenta, plays a critical role in determining the level of exposure of the fetus to maternal thyroid hormones.

Expression profiles of the three iodothyronine deiodinases, D1, D2, and D3, in the developing rat.

Thyroid hormone (TH) is essential for normal development in vertebrate species. Although the mechanisms by which TH regulates developmental processes are not fully understood, intracellular T3 levels are likely to be a critical aspect of the process. Furthermore, as different tissues and organs have specific temporal patterns of development, their T3 requirements may vary widely. Differential regulation of intracellular T3 levels in peripheral tissues as a result of differences in the activities of the three iodothyronine deiodinases (D1, D2, and D3) could offer an important means of achieving coordination of T3-dependent developmental processes among tissues. To obtain evidence for this concept we have documented the levels of expression of all three types of deiodinase in 11 tissues of the fetus, the neonate, and the adult rat. In most fetal tissues, D3 was the predominant deiodinase, but it declined after birth as the activities of D1 and D2 increased. Exceptions to this pattern were skin and brown adipose tissue (BAT), in which D2 activity was highest in the fetus, and testis and thyroid in which D2 activity was higher in the neonate than in the adult. D1 was the only 5'D enzyme expressed in liver, kidney and intestine at all stages studied, and D3 was not expressed in these tissues after birth. Thyroid, pituitary, and BAT expressed either D2 or D2 plus D1, but did not express D3 at any stage studied. Cerebrum, cerebellum, ovary, testis, skin, and placenta expressed all three deiodinases. Two other points were evident. First, the maximum 5'D activity attained, and thus presumably the amount of T3 generated, in liver, kidney, intestine, thyroid, pituitary, and BAT was very much higher than that in cerebrum, cerebellum, ovary, testis, skin, and placenta. Second, in the tissues where 5'D activity was relatively low, coexpression of D3 with D1 and D2 was the general rule, suggesting the need for very tight control of intracellular T3 levels. The findings are consistent with the view that the deiodinases play a major role in achieving the intracellular T3 levels that are optimal for the development of each tissue. Additional studies are in progress to demonstrate the functional consequences of these deiodinase expression patterns.

Isolation and characterization of the mouse gene for the type 3 iodothyronine deiodinase.

The type 3 iodothyronine deiodinase (D3) is a selenoenzyme that inactivates thyroid hormones by removing a iodine from the 5-position of the tyrosyl ring. D3 is highly expressed in many tissues during the early stages of development, and its activity is regulated by selected growth factors and various hormones. To gain further insights into the structure, functional role, and regulation of this enzyme, we screened a mouse liver genomic library with a rat D3 complementary DNA probe and isolated a 12-kb clone coding for the Dio3. Restriction analysis followed by Southern blotting and nucleotide sequencing demonstrated that the Dio3 contains a single exon, 1853 bp in length, that encodes the entire length of the messenger RNA expressed in murine placenta and neonatal skin. Primer extension experiments identified two potential transcriptional start sites located 77 and 60 nt upstream of the ATG translational start codon. The region immediately 5' to the start sites contains consensus TATA, CAAT, and GC elements. Furthermore, a 526-nucleotide genomic fragment from this region was demonstrated to efficiently drive a luciferase reporter construct when transfected into COS-7, XTC-2, or XL-2 cells or into primary cultures of rat preadipocytes derived from neonatal brown fat. In conclusion, D3 transcripts in the placenta and skin are encoded by the Dio3 gene from a single exon whose expression is regulated by an upstream region that contains several consensus promoter elements. Further characterization of this gene will provide new insights into the factors regulating the unique pattern of D3 expression during development.

Conserved cysteines in the type 1 deiodinase selenoprotein are not essential for catalytic activity.

The iodothyronine deiodinases are a family of oxidoreductases that catalyze the removal of iodide from thyroid hormones. Each of the three isoforms contain selenocysteine at its active site and several cysteine residues that may be important for catalytic activity. Of particular interest in the type I deiodinase (D1) is Cys124, which is vicinal to the selenocysteine at position 126, and Cys194, which has been conserved in all deiodinases identified to date. In the present studies, we have characterized the functional properties of C124A, C194A, and C124A/C194A D1 mutants, which were prepared by site-directed mutagenesis and expressed in COS-7 cells. In broken cell preparations, the sensitivity of the mutants to the selective D1 inhibitors propylthiouracil and aurothioglucose were unaltered. Mutagenesis at the Cys124 position was associated with a 7-11-fold increase in the Km of dithiothreitol, whereas Vmax values remained largely unchanged. However, both mutations resulted in marked decreases in Vmax values when glutathione or a reconstituted thioredoxin cofactor system were used in the assay. In contrast to the results of these in vitro studies, no impairment in deiodinating capability was noted in intact cells expressing equivalent levels of the mutant constructs. These studies demonstrate that Cys124 and Cys194 influence the reactivity of the D1 with thiol cofactors in in vitro assay systems but are not determinants of the sensitivity of the enzyme to propylthiouracil and aurothioglucose. Furthermore, the observation that the cysteine mutants are fully active in intact cells demonstrates that the results of commonly used broken cell assays do not accurately predict the activity of the D1 in intact cells and suggests that glutathione and thioredoxin are not the major thiols utilized in vivo to support D1 activity.

Transcriptional activation of type III inner ring deiodinase by growth factors in cultured rat brown adipocytes.

The activity of the type III inner ring deiodinase (DIII), which converts T4 and T3 to inactive metabolites, is induced by serum and growth factors in primary cultures of rat brown adipocytes. The contribution of pretranslational mechanisms to this increase in DIII activity was examined in the present studies. DIII mRNA is undetectable in differentiated brown adipocytes when cultured in serum-free medium. However, exposure to epidermal growth factor (EGF), acidic or basic fibroblast growth factors (aFGF or bFGF) increase DIII transcript levels. Lesser inductions are found with platelet-derived growth factor, and insulin-like growth factor I has no effect. Maximal induction of DIII mRNA is obtained after 9 h of exposure to EGF, bFGF, or aFGF at a concentration of 10 ng/ml. The increase in DIII mRNA in response to aFGF, bFGF, and EGF requires gene transcription and protein synthesis, as the inductive effect on mRNA is completely blocked by actinomycin D or cycloheximide. The DIII mRNA half-life is 4 h when stimulated with bFGF and increases to 12 h when 10% serum, EGF, or aFGF is present. In conclusion, EGF, aFGF, and bFGF increase DIII mRNA expression in differentiated brown adipocytes. This effect appears to be exerted at the level of both enhanced transcription and mRNA stabilization.

Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms.

The type 2 5'-deiodinase (D2) appears to play an important role in maintaining the intracerebral T3 content relatively constant during changes in thyroidal state. Previous studies have demonstrated that the regulation of this enzyme by thyroid hormone and its analogs occurs at a posttranslational level. The availability of the rat D2 complementary DNA now allows an assessment of whether pretranslational regulation of this enzyme also occurs in the cerebral cortex. In rats rendered hypothyroid by the addition of methimazole to the drinking water, D2 messenger RNA (mRNA) is increased 70% (P = 0.03). Treatment with L-T3 (50 microg/100 g BW) for 4 days results in an 80% decrease in D2 mRNA compared with that in euthyroid controls (P < 0.001). Administration of lower doses of L-T3 (0.25-3 microg/100 g BW x day) is associated with a dose-dependent decrease in cortical D2 mRNA, but little or no change in D2 activity. The decrease in D2 mRNA in response to T3 treatment can be demonstrated within 4 h. Treatment of hypothyroid rats for 2 weeks with graded doses of L-T4 (0.1-1.5 microg/100 g BW x day) results in a significant decrease in cortical D2 activity, but not mRNA. The association between D2 activity and D2 mRNA in euthyroid, hypothyroid, and hormone-treated rats across a full range of thyroidal states suggests that L-T4 treatment is associated with greater changes in cortical D2 activity (via posttranslational effects) than mRNA, whereas L-T3 treatment has a greater effect on decreasing D2 mRNA (i.e. pretranslational effects). In conclusion, these studies demonstrate both pre- and posttranslational regulation of cortical D2 expression. The relative contribution of each mechanism depends on the ambient thyroid hormone concentration.

The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain.

Thyroid hormone plays an essential role in mammalian brain maturation and function, in large part by regulating the expression of specific neuronal genes. In this tissue, the type 2 deiodinase (D2) appears to be essential for providing adequate levels of the active thyroid hormone 3,5,3'-triiodothyronine (T3) during the developmental period. We have studied the regional and cellular localization of D2 mRNA in the brain of 15-day-old neonatal rats. D2 is expressed in the cerebral cortex, olfactory bulb, hippocampus, caudate, thalamus, hypothalamus, and cerebellum and was absent from the white matter. At the cellular level, D2 is expressed predominantly, if not exclusively, in astrocytes and in the tanycytes lining the third ventricle and present in the median eminence. These results suggest a close metabolic coupling between subsets of glial cells and neurons, whereby thyroxine is taken up from the blood and/or cerebrospinal fluid by astrocytes and tanycytes, is deiodinated to T3, and then is released for utilization by neurons.

The deiodinase family of selenoproteins.

The realization some forty years ago that several iodothyronine compounds are present in the circulation suggested that deiodination occurs in various tissues. Subsequently, deiodination was indeed documented in in vivo studies. Later, using in vitro assay techniques, three deiodinase processes, termed types 1, 2 and 3, were defined that differed in terms of tissue distribution, reaction kinetics, efficiency of substrate utilization and sensitivity to inhibitors. Although purification of the deiodinase enzymes has continued to be problematic, recent molecular cloning studies have identified cDNAs for these three deiodinase isoforms from multiple species. These cDNAs have provided important insights into the structural characteristics of this family of enzymes. Foremost among the structural features has been the demonstration that all three deiodinase isoforms contain at their active site the uncommon amino acid selenocysteine which is of critical importance to their catalytic activity. The availability of cDNAs for these enzymes provides important reagents for pursuing additional studies aimed at defining their biochemical features and roles in thyroid hormone economy.

Expression of the type II iodothyronine deiodinase in cultured rat astrocytes is selenium-dependent.

The iodothyronine deiodinases are a family of selenoproteins that metabolize thyroxine and other thyroid hormones to active and inactive metabolites in a number of tissues including brain. Using primary cultures of rat astroglial cells as a model system, we demonstrate that the mRNA for the type II iodothyronine deiodinase (DII) selenoenzyme is rapidly and markedly induced by forskolin and 8-bromo-cAMP. The induction of DII activity, however, was significantly impaired by culturing cells in selenium-deficient medium for 7 days. Under such conditions, the addition of selenium resulted in a rapid increase in cAMP-induced DII activity that was dose-dependent, with maximal effects noted within 2 h. Cycloheximide blocked this effect of selenium on restoring cAMP-induced DII activity, whereas actinomycin D did not. These data demonstrate that the DII selenoenzyme is expressed in cultured astrocytes and that the induction of DII activity by cAMP analogues appears to be mediated, at least in part, by pretranslational mechanisms. Furthermore, selenium deprivation impairs the expression of DII activity at the level of translation.

Cloning of the mammalian type II iodothyronine deiodinase. A selenoprotein differentially expressed and regulated in human and rat brain and other tissues.

The deiodination of thyroid hormones in extrathyroidal tissues plays an important role in modulating thyroid hormone action. The type II deiodinase (DII) converts thyroxine to the active hormone 3,5,3'-triiodothyronine, and in the rat is expressed in the brain, pituitary gland, and brown adipose tissue (BAT). Complementary DNAs (cDNAs) for the types I and III deiodinases (DI and DIII, respectively) have been isolated and shown to code for selenoproteins. However, information concerning the structure of the mammalian DII remains limited, and the pattern of its expression in human tissues is undefined. We report herein the identification and characterization of rat and human DII cDNAs. Both code for selenoproteins and exhibit limited regions of homology with the DI and DIII. In the rat pituitary and BAT, DII mRNA levels are altered more than 10-fold by changes in the thyroid hormone status of the animal. Northern analysis of RNA derived from human tissues reveals expression of DII transcripts in heart, skeletal muscle, placenta, fetal brain, and several regions of the adult brain. These studies demonstrate that: (a) the rat and human DII are selenoproteins, (b) DII expression in the rat is regulated, at least in part, at the pretranslational level in some tissues, and (c) DII is likely to be of considerable physiologic importance in thyroid hormone economy in the human fetus and adult.

Cloning of a cDNA for the type II iodothyronine deiodinase.

Three types of iodothyronine deiodinase have been identified in vertebrate tissues. cDNAs for the types I and III have been cloned and shown to contain an inframe TGA that codes for selenocysteine at the active site of the enzyme. We now report the cloning of a cDNA for a type II deiodinase using a reverse transcription/polymerase chain reaction strategy and RNA obtained from Rana catesbeiana tissues. This cDNA (RC5'DII) manifests limited but significant homology with other deiodinase cDNAs and contains a conserved in-frame TGA codon. Injection of capped in vitro synthesized transcripts of the cDNA into Xenopus laevis oocytes results in the induction of deiodinase activity with characteristics typical of a type II deiodinase. The levels of RC5'DII transcripts in R. catesbeiana tadpole tail and liver mRNA at stages XII and XXIII correspond well with that of type II deiodinase activity but not that of the type III activity in these tissues. These findings indicate that the amphibian type II 5'-deiodinase is a structurally unique member of the family of selenocysteine-containing deiodinases.