Valerie Anne Galton

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.

Type 3 deiodinase is critical for the maturation and function of the thyroid axis

Developmental exposure to appropriate levels of thyroid hormones (THs) in a timely manner is critical to normal development in vertebrates. Among the factors potentially affecting perinatal exposure of tissues to THs is type 3 deiodinase (D3). This enzyme degrades THs and is highly expressed in the pregnant uterus, placenta, and fetal and neonatal tissues. To determine the physiological role of D3, we have generated a mouse D3 knockout model (D3KO) by a targeted inactivating mutation of the Dio3 gene in mouse ES cells. Early in life, D3KO mice exhibit delayed 3,5,3-triiodothyronine (T3) clearance, a markedly elevated serum T3 level, and overexpression of T3-inducible genes in the brain. From postnatal day 15 to adulthood, D3KO mice demonstrate central hypothyroidism, with low serum levels of 3,5,3,5-tetraiodothyronine (T4) and T3, and modest or no increase in thyroid-stimulating hormone (TSH) concentration. Peripheral tissues are also hypothyroid. Hypothalamic T3 content is decreased while thyrotropin-releasing hormone (TRH) expression is elevated. Our results demonstrate that the lack of D3 function results in neonatal thyrotoxicosis followed later by central hypothyroidism that persists throughout life. These mice provide a new model of central hypothyroidism and reveal a critical role for D3 in the maturation and function of the thyroid axis.

Defining the Roles of the Iodothyronine Deiodinases: Current Concepts and Challenges

As is typical of other hormone systems, the actions of the thyroid hormones (TH) differ from tissue to tissue depending upon a number of variables. In addition to varying expression levels of TH receptors and transporters, differing patterns of TH metabolism provide a critical mechanism whereby TH action can be individualized in cells depending on the needs of the organism. The iodothyronine deiodinases constitute a family of selenoenzymes that selectively remove iodide from thyroxine and its derivatives, thus activating or inactivating these hormones. Three deiodinases have been identified, and much has been learned regarding the differing structures, catalytic activities, and expression patterns of these proteins. Because of their differing properties, the deiodinases appear to serve varying functions that are important in regulating metabolic processes, TH action during development, and feedback control of the thyroid axis. This review will briefly assess these functional roles and others proposed for the deiodinases and examine some of the current challenges in expanding our knowledge of these important components of the thyroid homeostatic system.

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 in-frame 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 Xenopuslaevis 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.

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.

A Protective Role for Type 3 Deiodinase, a Thyroid Hormone-Inactivating Enzyme, in Cochlear Development and Auditory Function

Thyroid hormone is necessary for cochlear development and auditory function, but the factors that control these processes are poorly understood. Previous evidence indicated that in mice, the serum supply of thyroid hormone is augmented within the cochlea itself by type 2 deiodinase, which amplifies the level of T(3), the active form of thyroid hormone, before the onset of hearing. We now report that type 3 deiodinase, a thyroid hormone-inactivating enzyme encoded by Dio3, is expressed in the immature cochlea before type 2 deiodinase. Dio3-/- mice display auditory deficits and accelerated cochlear differentiation, contrasting with the retardation caused by deletion of type 2 deiodinase. The Dio3 mRNA expression pattern in the greater epithelial ridge, stria vascularis, and spiral ganglion partly overlaps with that of thyroid hormone receptor beta (TRbeta), the T(3) receptor that is primarily responsible for auditory development. The proposal that type 3 deiodinase prevents premature stimulation of TRbeta was supported by deleting TRbeta, which converted the Dio3-/- cochlear phenotype from one of accelerated to one of delayed differentiation. The results indicate a protective role for type 3 deiodinase in hearing. The auditory system illustrates the considerable extent to which tissues can autoregulate their developmental response to thyroid hormone through both type 2 and 3 deiodinases.

Life without Thyroxine to 3,5,3′-Triiodothyronine Conversion: Studies in Mice Devoid of the 5′-Deiodinases

Considerable indirect evidence suggests that the type 2 deiodinase (D2) generates T(3) from T(4) for local use in specific tissues including pituitary, brown fat, and brain, whereas the type I deiodinase (D1) generates T(3) from T(4) in the thyroid and peripheral tissues primarily for export to plasma. From studies in deiodinase-deficient mice, the importance of the D2 for local T(3) generation has been confirmed. However, the phenotypes of these D1 knockout (KO) and D2KO mice are surprisingly mild and their serum T(3) level, general health, and reproductive capacity are unimpaired. To explore further the importance of 5deiodination in thyroid hormone economy, we used a mouse devoid of both D1 and D2 activity. In general, the phenotype of the D1/D2KO mouse is the sum of the phenotypes of the D1KO and D2KO mice. It appears healthy and breeds well, and most surprisingly its serum T(3) level is normal. However, impairments in brain gene expression and possibly neurological function are somewhat greater than those seen in the D2KO mouse, and the serum rT(3) level is elevated 6-fold in the D1/D2KO mouse but only 2-fold in the D1KO mouse and not at all in the D2KO mouse. The data suggest that whereas D1 and D2 are not essential for the maintenance of the serum T(3) level, they do serve important roles in thyroid hormone homeostasis, the D2 being critical for local T(3) production and the D1 playing an important role in iodide conservation by serving as a scavenger enzyme in peripheral tissues and the thyroid.

Optimal bone strength and mineralization requires the type 2 iodothyronine deiodinase in osteoblasts

Hypothyroidism and thyrotoxicosis are each associated with an increased risk of fracture. Although thyroxine (T4) is the predominant circulating thyroid hormone, target cell responses are determined by local intracellular availability of the active hormone 3,5,3′-L-triiodothyronine (T3), which is generated from T4 by the type 2 deiodinase enzyme (D2). To investigate the role of locally produced T3 in bone, we characterized mice deficient in D2 (D2KO) in which the serum T3 level is normal. Bones from adult D2KO mice have reduced toughness and are brittle, displaying an increased susceptibility to fracture. This phenotype is characterized by a 50% reduction in bone formation and a generalized increase in skeletal mineralization resulting from a local deficiency of T3 in osteoblasts. These data reveal an essential role for D2 in osteoblasts in the optimization of bone strength and mineralization.

Thyroid Hormone-Regulated Mouse Cerebral Cortex Genes Are Differentially Dependent on the Source of the Hormone: A Study in Monocarboxylate Transporter-8- and Deiodinase-2-Deficient Mice

Thyroid hormones influence brain development through the control of gene expression. The concentration of the active hormone T(3) in the brain depends on T(3) transport through the blood-brain barrier, mediated in part by the monocarboxylate transporter 8 (Mct8/MCT8) and the activity of type 2 deiodinase (D2) generating T(3) from T(4). The relative roles of each of these pathways in the regulation of brain gene expression is not known. To shed light on this question, we analyzed thyroid hormone-dependent gene expression in the cerebral cortex of mice with inactivated Mct8 (Slc16a2) and Dio2 genes, alone or in combination. We used 34 target genes identified to be controlled by thyroid hormone in microarray comparisons of cerebral cortex from wild-type control and hypothyroid mice on postnatal d 21. Inactivation of the Mct8 gene (Mct8KO) was without effect on the expression of 31 of these genes. Normal gene expression in the absence of the transporter was mostly due to D2 activity because the combined disruption of Mct8 and Dio2 led to similar effects as hypothyroidism on the expression of 24 genes. Dio2 disruption alone did not affect the expression of positively regulated genes, but, as in hypothyroidism, it increased that of negatively regulated genes. We conclude that gene expression in the Mct8KO cerebral cortex is compensated in part by D2-dependent mechanisms. Intriguingly, positive or negative regulation of genes by thyroid hormone is sensitive to the source of T(3) because Dio2 inactivation selectively affects the expression of negatively regulated genes.

American Thyroid Association Guide to investigating thyroid hormone economy and action in rodent and cell models.

BACKGROUNDnAn in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasis is critical for the development of new diagnostic and treatment approaches for patients with thyroid disease.nnnSUMMARYnImportant clinical practices in use today for the treatment of patients with hypothyroidism, hyperthyroidism, or thyroid cancer are the result of laboratory discoveries made by scientists investigating the most basic aspects of thyroid structure and molecular biology. In this document, a panel of experts commissioned by the American Thyroid Association makes a series of recommendations related to the study of thyroid hormone economy and action. These recommendations are intended to promote standardization of study design, which should in turn increase the comparability and reproducibility of experimental findings.nnnCONCLUSIONSnIt is expected that adherence to these recommendations by investigators in the field will facilitate progress towards a better understanding of the thyroid gland and thyroid hormone dependent processes.