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Title: Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action

Abstract

Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors {alpha} (TR{alpha}) and {beta}A (TR{beta}A) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TR{alpha} gene expression whereas a marked up-regulation of TR{beta}A mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TR{beta}A mRNA in head and tail tissue within 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TR{beta}A mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TR{beta}A gene expression analysis for detection of agonistic and antagonistic effects on T3more » action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TR{beta}A expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TR{beta}A mRNA up-regulation. Results of this study suggest that TR{beta}A mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.« less

Authors:
 [1];  [2];  [3];  [3];  [2];  [4]
  1. Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Mueggelseedamm 301, Berlin D-12587 (Germany). E-mail: r.opitz@igb-berlin.de
  2. Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Mueggelseedamm 301, Berlin D-12587 (Germany)
  3. Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143-2280 (United States)
  4. (Germany)
Publication Date:
OSTI Identifier:
20783448
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 212; Journal Issue: 1; Other Information: DOI: 10.1016/j.taap.2005.06.014; PII: S0041-008X(05)00377-7; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMPHIBIANS; BIOLOGICAL MODELS; GENES; IN VIVO; LARVAE; LIMBS; METAMORPHOSIS; POLYMERASE CHAIN REACTION; RECEPTORS; THYROID; THYROXINE; TRIIODOTHYRONINE

Citation Formats

Opitz, Robert, Lutz, Ilka, Nguyen, Ngoc-Ha, Scanlan, Thomas S., Kloas, Werner, and Department of Endocrinology, Institute of Biology, Humboldt University Berlin, D-10115 Berlin. Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action. United States: N. p., 2006. Web. doi:10.1016/j.taap.2005.06.014.
Opitz, Robert, Lutz, Ilka, Nguyen, Ngoc-Ha, Scanlan, Thomas S., Kloas, Werner, & Department of Endocrinology, Institute of Biology, Humboldt University Berlin, D-10115 Berlin. Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action. United States. doi:10.1016/j.taap.2005.06.014.
Opitz, Robert, Lutz, Ilka, Nguyen, Ngoc-Ha, Scanlan, Thomas S., Kloas, Werner, and Department of Endocrinology, Institute of Biology, Humboldt University Berlin, D-10115 Berlin. Sat . "Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action". United States. doi:10.1016/j.taap.2005.06.014.
@article{osti_20783448,
title = {Analysis of thyroid hormone receptor {beta}A mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action},
author = {Opitz, Robert and Lutz, Ilka and Nguyen, Ngoc-Ha and Scanlan, Thomas S. and Kloas, Werner and Department of Endocrinology, Institute of Biology, Humboldt University Berlin, D-10115 Berlin},
abstractNote = {Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors {alpha} (TR{alpha}) and {beta}A (TR{beta}A) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TR{alpha} gene expression whereas a marked up-regulation of TR{beta}A mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TR{beta}A mRNA in head and tail tissue within 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TR{beta}A mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TR{beta}A gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TR{beta}A expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TR{beta}A mRNA up-regulation. Results of this study suggest that TR{beta}A mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.},
doi = {10.1016/j.taap.2005.06.014},
journal = {Toxicology and Applied Pharmacology},
number = 1,
volume = 212,
place = {United States},
year = {Sat Apr 01 00:00:00 EST 2006},
month = {Sat Apr 01 00:00:00 EST 2006}
}
  • The expression of several neurotransmitter and drug receptors from injected exogenous mRNA in Xenopus laevis oocytes has been demonstrated by electrophysiological measurements of ion channel activation. The expression of specific receptors for peptide hormones in such a translation system would facilitate studies on the structure and regulation of cell-surface receptors as well as their coupling to membrane transduction mechanisms. The expression of receptors for calcium-mobilizing hormones in Xenopus oocytes was sought by analysis of phospholipid turnover in hormone-stimulated oocytes. For this purpose, Xenopus oocytes were injected with mRNA extracted from bovine adrenal and pituitary glands and incubated with myo-(/sup 3/H)inositolmore » to label plasma-membrane phosphatidylinositol phosphates. The expression of functionally active receptors for angiotensin II (AII) and thyrotropin-releasing hormone (TRH) was demonstrated by the stimulation of (/sup 3/H)inositol phosphate production by AII and TRH in the mRNA-injected, (/sup 3/H)inositol-prelabeled oocytes. The ability of AII and TRH to act by way of newly synthesized receptors from mammalian endocrine tissues to stimulate phosphatidylinositol polyphosphate hydrolysis in Xenopus oocytes suggests a generalized and conserved mechanism of receptor coupling to the transduction mechanism responsible for activation of phospholipase C in the plasma membrane.« less
  • The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNAmore » in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.« less
  • The cytotoxic effects of sodium selenite on developing tadpoles (Xenopus laevis) were examined by scanning, light, and electron microscopy. Selenium exposure resulted in disorganization, vacuolization, and swelling of the outer layer of epithelial cells in the tadpole epidermis. Examination of muscle cells in the somites revealed myofibril disorganization and cell degeneration. Mitochondria in both epithelial and muscle cells were swollen and showed loss of cristae. It is likely that sublethal exposures to selenium compounds result in cellular damage which could affect motility, and thus survival, over longer periods of time.
  • Tadpoles of Xenopus laevis in a small water phantom were exposed to doses of 500, 1000, 1500, 2000, and 2500 r high-energy electrons from a 31-Mev betatron. Investigations were made concerning the infiuence of dose-rate, total dose, and site of the irradiated animals in the phartom on lethality. The experiments were repeated after one year. Data are tabulated. After a single dose of 500 r the lethality of the tadpoles was considerably higher at a dose rate of 300 r/min than at 50 r/min. The lethality of the animals increased with the depth of the phantom and also when themore » dose and dose-rate were constant. This phenomenon was attributed to rapid changes in linear-energy transfer with increasing depth in water.« less
  • Mutations in the gene encoding human thyroid hormone receptor {beta}(hTR{beta}) have been associated with generalized resistance to thyroid hormone (GRTH). This disorder is associated with significant behavoral abnormalities. We examined the hTR{beta} gene in a family with members who manifest inappropriately normal TSH, elevated free T{sub 4}, and free and total T{sub 3}. Sequence analysis showed a cytosine to thymine transition at nucleotide 1642 in one allele of the index patient`s genomic DNA. This altered proline to serine at codon 453. The resulting mutant receptor when expressed in vitro bound DNA with high affinity, but the T{sub 3} affinity ofmore » the receptor was impaired. The mutant TR demonstrated a dominant negative effect when cotransfected with two isoforms of wild-type receptor and also in the presence of TR variant {alpha}2 in COS-1 cells. Mutations of codon 453 occur more frequently than at other sites, and four different amino acid substitutions have been reported. Significant differences in phenotype occur among affected individuals, varying from normality to moderately severe GRTH. There is no clear correlation between K{sub a} or in vitro function of the mutant receptor, and phenotype. This study extends the association between GRTH and illness, and indicates that early diagnosis and counseling are needed in families with TR{beta}1 abnormalities. 34 refs., 5 figs., 2 tabs.« less