Serge Van der Geyten

Expression of Chicken Hepatic Type I and Type III Iodothyronine Deiodinases during Embryonic Development

In embryonic chicken liver (ECL) two types of iodothyronine deiodinases are expressed: D1 and D3. D1 catalyzes the activation as well as the inactivation of thyroid hormone by outer and inner ring deiodination, respectively. D3 only catalyzes inner ring deiodination. D1 and D3 have been cloned from mammals and amphibians and shown to contain a selenocysteine (Sec) residue. We characterized chicken D1 and D3 complementary DNAs (cDNAs) and studied the expression of hepatic D1 and D3 messenger RNAs (mRNAs) during embryonic development. Oligonucleotides based on two amino acid sequences strongly conserved in the different deiodinases (NFGSCTSecP and YIEEAH) were used for reverse transcription-PCR of poly(A 1 ) RNA isolated from embryonic day 17 (E17) chicken liver, resulting in the amplification of two 117-bp DNA fragments. Screening of an E17 chicken liver cDNA library with these probes led to the isolation of two cDNA clones, ECL1711 and ECL1715. The ECL1711 clone was 1360 bp long and lacked a translation start site. Sequence alignment showed that it shared highest sequence identity with D1s from other vertebrates and that the coding sequence probably lacked the first five nucleotides. An ATG start codon was engineered by site-directed mutagenesis, generating a mutant (ECL1711M) with four additional codons (coding for MGTR). The open reading frame of ECL1711M coded for a 249-amino acid protein showing 58 ‐ 62% identity with mammalian D1s. An in-frame TGA codon was located at position 127, which is translated as Sec in the presence of a Sec insertion sequence (SECIS) identified in the 39-untranslated region. Enzyme activity expressed in COS-1 cells by transfection with ECL1711M showed the same catalytic, substrate, and inhibitor specificities as native chicken D1. The ECL1715 clone was 1366 bp long and also lacked a translation start site. Sequence alignment showed that it was most homologous with D3 from other species and that the coding sequence lacked approximately the first 46 nucleotides. The deduced amino acid sequence showed 62‐72% identity with the D3 sequences from other species, including a putative Sec residue at a corresponding position. The 39-untranslated region of ECL1715 also contained a SECIS element. These results indicate that ECL1711 and ECL1715 are nearfull-length cDNA clones for chicken D1 and D3 selenoproteins, respectively. The ontogeny of D1 and D3 expression in chicken liver was studied between E14 and 1 day after hatching (C1). D1 activity showed a gradual increase from E14 until C1, whereas D1 mRNA level remained relatively constant. D3 activity and mRNA level were highly significantly correlated, showing an increase from E14 to E17 and a strong decrease thereafter. These results suggest that the regulation of chicken hepatic D3 expression during embryonic development occurs predominantly at the pretranslational level. (Endocrinology 138: 5144 ‐5152, 1997)

Characterization of a propylthiouracil-insensitive type I iodothyronine deiodinase

Mammalian type I iodothyronine deiodinase (D1) activates and inactivates thyroid hormone by outer ring deiodination (ORD) and inner ring deiodination (IRD), respectively, and is potently inhibited by propylthiouracil (PTU). Here we describe the cloning and characterization of a complementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreochromis niloticus, tilapia). This complementary DNA codes for a protein of 248 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 126. The 39 untranslated region contains two putative Sec insertion sequence (SECIS) elements. Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as native tilapia D1 (tD1), i.e. rT3 . . T4 . T3 sulfate . T3. Native and recombinant tD1 show equally low sensitivities to inhibition by PTU, iodoacetate, and gold thioglucose compared with the potent inhibitions observed with mammalian D1s. Because the residue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased ORD and somewhat increased IRD activity, but was still insensitive to PTU. These results provide new information about the structure-activity relationship of D1 concerning two characteristic properties, i.e. catalysis of both ORD and IRD, and inhibition by PTU. (Endocrinology 138: 5153‐5160, 1997)

Type 2 Iodothyronine Deiodinase Is Essential for Thyroid Hormone-Dependent Embryonic Development and Pigmentation in Zebrafish

Despite the known importance of thyroid hormones (THs) in vertebrate growth and development, the role of tissue-specific TH activation in early embryogenesis remains unclear. We therefore examined the function of type 2 iodothyronine deiodinase (D2), one of the two tissue-specific enzymes catalyzing the conversion of T4 to T3, in developing zebrafish embryos (Danio rerio). Microinjection of early embryos with antisense oligonucleotides targeting either the D2 translation start site or the splice junction between the first exon and intron induced delays in development and pigmentation, as determined through the measurement of otic vesicle length, head-trunk angle, and pigmentation index at 31 h after fertilization. The antisense-induced delays in developmental progression and pigmentation were reversible through treatment with T3, suggesting that these phenotypic effects may be due to the depletion of intracellular T3 levels. Additional evidence for this hypothesis was provided by quantitative RT-PCR analysis of TH receptor-beta expression in D2 knockdown embryos, revealing a significant down-regulation of this T3-induced transcript that could be reversed by T3 treatment. Tyrosinase expression was also down-regulated in D2 knockdown embryos to a greater degree than could be predicted by the observed delay in developmental progression, suggesting that reduced D2 activity and resultant low intracellular T3 availability may directly influence pigmentation in zebrafish. These data indicate that TH activation by D2 is essential for embryonic development and pigmentation in zebrafish.

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.

Inhibition and activation of the thyroidal axis by the adrenal axis in vertebrates.

Hormones of the adrenal or interrenal axis and stress situations which induce elevated glucocorticoid plasma levels (e.g. handling and starvation), inhibit thyroid function in growing and adult vertebrates. However, data indicate that during foetal and embryonic development (mammals and birds) or during larval growth and metamorphosis (fish and amphibians), the adrenal axis may stimulate thyroid function. Recent findings have provided some information concerning this stimulatory interference of the adrenal axis. In amphibians corticotropin releasing hormone and not thyrotropin releasing hormone is thyrotropic during metamorphosis, thus providing the substrate T4 necessary for T3 production. Other data indicate that the increase in plasma T3 at metamorphic climax may be the result of an inhibition of the T3 degrading activity, rather than stimulation of the T4 into T3 converting activity, and that glucocorticoids may be responsible for this. Also, in the chick embryo glucocorticoids effectively increase plasma T3 concentration by reducing the hepatic T3 degrading activity, whereas corticotropin releasing hormone also induces an elevation in the thyrotropin plasma levels and hence raises T4 concentrations which may function as a substrate for T3 production.

Cloning and Characterization of Type III Iodothyronine Deiodinase from the Fish Oreochromis niloticus

Type III iodothyronine deiodinase (D3) catalyzes the inner ring deiodination (IRD) of T4 and T3 to the inactive metabolites rT3 and 3,3′-diiodothyronine (3,3′-T2), respectively. Here we describe the cloning and characterization of complementary DNA (cDNA) coding for D3 in fish (Oreochromis niloticus, tilapia). This cDNA contains 1478 nucleotides and codes for a protein of 267 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 131. The deduced amino acid sequence shows 57–67% identity with frog, chicken, and mammalian D3, 33–39% identity with frog, fish (Fundulus heteroclitus) and mammalian D2, and 30–35% identity with fish (tilapia), chicken, and mammalian D1. The 3′ UTR contains a putative Sec insertion sequence (SECIS) element. Recombinant tilapia D3 (tD3) expressed in COS-1 cells and native tD3 in tilapia brain microsomes show identical catalytic activities, with a strong preference for IRD of T3 (Km ∼20 nm). IRD of [3,5-125I]T3 by native and recombina...

Effects of dexamethasone treatment on iodothyronine deiodinase activities and on metamorphosis-related morphological changes in the axolotl (Ambystoma mexicanum).

In amphibians, there is a close interaction between the interrenal and the thyroidal axes. Hypothalamic corticotropin-releasing hormone or related peptides stimulate thyroidal activity by increasing thyrotropin synthesis and release, while corticosterone accelerates both spontaneous and thyroid hormone-induced metamorphosis. One of the mechanisms that is thought to contribute to this acceleration is a corticosterone-induced change in peripheral deiodinating activity. The present experiments were designed to investigate further the effects of glucocorticoid treatment on amphibian deiodinase activities and to explore the possible role of these effects in metamorphosis. Neotenic axolotls (Ambystoma mexicanum) were treated either acutely or chronically with dexamethasone (DEX) and changes in type II and type III iodothyronine deiodinase (D2 and D3) activities were studied in liver, kidney, and brain. In addition, gill length, tail height, and body weight were measured at regular intervals in the chronically treated animals in search of metamorphosis-related changes. A single injection of 50 microg DEX decreased hepatic D3 activity (6-48 h) while it increased D2 activity in brain (6-48 h) and to a lesser extent in kidney (24 h). These changes were accompanied by an increase in plasma T(3) levels (48 h). Samples taken during chronic treatment with 20 or 100 microg DEX showed that both hepatic D2 and D3 activities were decreased on day 26, while renal D3 activity was decreased but only in the 20 microg dose group. All other deiodinase activities were not different from those in control animals. At 25 days, all DEX-treated axolotls showed a clear reduction in gill length, tail height, and body weight, changes typical of metamorphosis. Prolongation of the treatment up to 48 days resulted in complete gill resorption by days 44-60. Although probably several mechanisms contribute to these DEX-induced metamorphic changes, the interaction with thyroid function via a sustained downregulation of hepatic D3 may be one of them.

Type I iodothyronine deiodinase in euthyroid and hypothyroid chicken cerebellum.

Immunocytochemistry using polyclonal anti-type I deiodinase (D1) led to the localization of D1 protein in the internal granule cells of the cerebellum in 1-day-old chicks, which was confirmed by the presence of in vitro D1 activity. Western blot analysis of hepatic and cerebellar extracts revealed a band of 27 kDa. In hypothyroid embryos D1 was expressed in both the internal and external granule cell layer and the signal diminished with more severe hypothyroidism, which is in agreement with the expected downregulation of D1 activity during hypothyroidism. In accordance with the protein data, hypothyroidism resulted in the downregulation of cerebellar D1 mRNA. Finally, histological stainings confirmed that the lack of staining in the external germinal layer of 1-day-old euthyroid chicks was due to the fact that migration of immature granule cells from the external towards the internal layer was completed at this stage while cell migration was retarded in hypothyroid animals.

Regulation of Thyroid Hormone Availability by Iodothyronine Deiodinases at the Blood‐Brain Barrier in Birds

Abstract: It is accepted that type II iodothyronine deiodinase (D2) is predominantly found in brain, where it maintains homeostasis of thyroid hormone (TH) levels. The current study describes the production of a polyclonal D2 antiserum and its use in the comparison of D2 protein distribution with that of type I (D1) and type III (D3) deiodinase protein in the chicken choroid plexus (CP). Immunocytochemistry showed high D2 protein expression in the epithelial cells of the CP, whereas the D1 and D3 proteins were absent. Furthermore, dexamethasone treatment led to an upregulation of the D2 protein in these cells.