Severe Resistance to Thyroid Hormone Beta in a Patient with Athyreosis.

We report a patient with congenital hypothyroidism due to athyreosis complicated by a heterozygous thyroid hormone receptor beta (THRß) gene mutation (R320L), resulting in a severe resistance to thyroid hormone beta phenotype. The proband inherited the mutant allele from his father, presenting a very mild phenotype. While the precise reason for this discrepancy remains unknown, we postulate the possibility of de novo mutation and mosaicism in the father. Correlating thyrotropin (TSH) with free thyroxine (fT4) allowed us to predict the amount of fT4 required to normalize the proband's TSH, which supported the treatment with high dose of levothyroxine.

Novel Mutation (T273R) in Thyroid Hormone Receptor ß Gene Provides Further Insight into Cryptic Negative Regulation by Thyroid Hormone.

Production of thyroid hormone is precisely regulated in a negative feed-back mechanism that depends critically on thyroid hormone receptor ß (TRß). This mechanism decreases production of thyrotropin- releasing hormone (TRH) and thyrotropin (TSH) in the hypothalamus and pituitary gland in response to high levels of circulating thyroid hormones (TH). Despite the wealth of accumulated knowledge, it is still not clear how exactly this negative regulation is executed. The syndrome of resistance to thyroid hormone (RTH), in which the levels of TH are not properly sensed, represents naturally occurring situations in which molecular components of this regulation are displayed and may be uncovered. TRß, which is central to this regulation, is in the majority of RTH cases mutated in a way that preserves some functions of the receptor. Approximately 150 different mutations in TRß have been identified to date. Here, we hypothesized that additional pathogenic mutations in TRß are likely to exist in human population and analysed clinical cases with suspected RTH. In keeping with our prediction, analysis of 17 patients from nine families led to identification of four presumed pathogenic mutations of TRß, including a previously unknown mutation, T273R. This suggests that threonine 273 is likely to be critical for the normal function of TRß, possibly due to its role in helix 12 mobility and interaction with coactivators, and thus supports the concept that TRß-dependent trans-activating function is necessary for the inhibition of TRH and TSH expression in response to elevated levels of TH.

An investigation into the effects of maternal supplementation with excess iodine on the mechanisms and impacts of reduced IgG absorption in the lamb postpartum.

An experiment was conducted to determine: (1) the effect of excess maternal I supplementation on the thyroid hormone status of the ewe and her progeny; (2) potential mechanisms underpinning the failure of passive transfer associated with excess I and (3) the growing lambs' response to natural gastrointestinal infection. Twin-bearing ewes received one of two treatments (n 32/treatment group): basal diet (C) or C plus 26·6 mg of iodine/ewe per d (I), supplied as calcium iodate. Ewes were individually fed from day 119 of gestation to parturition. Progeny of I ewes had lower (P<0·01) serum IgG concentrations from 24 h to 28 d postpartum but higher serum IgG concentrations at day 70 postpartum (P<0·05). I supplementation increased the relative expression of Fc receptor, IgA, IgM high affinity and polymeric Ig receptor in the ileum of the lamb at 24 h postpartum; however, thyroid hormone receptor-ß (THRB) and ß-2-microglobulin (B2M) expression declined (P<0·05). Progeny of I ewes had higher growth rates to weaning (P<0·05) and lower faecal egg count (FEC) for Nematodirus battus (P<0·05) between weeks 6 and 10 postpartum. In conclusion, excess maternal I supplementation negatively affected the thyroid hormone status, serum IgG concentration, ileal morphology and the gene expression of THRB and B2M in the ileum and ras-related protein (RAB) RAB25 and the mucin gene (MUC) MUC1 in the duodenum of the lamb postpartum. These effects were followed by an enhancement of average daily gain and lower N. battus FEC in the pre-weaning period of I-supplemented lambs.

Successful every-other-day liothyronine therapy for severe resistance to thyroid hormone beta with a novel THRB mutation; case report.

BACKGROUND: Resistance to thyroid hormone beta (RTHß) is a rare and usually dominantly inherited syndrome caused by mutations of the thyroid hormone receptor ß gene (THRB). In severe cases, it is rarely challenging to control manifestations using daily therapeutic replacement of thyroid hormone. CASE PRESENTATION: The present case study concerns an 8-year-old Japanese girl with a severe phenotype of RTH (TSH, fT3, and fT4 were 34.0 mU/L, >25.0 pg/mL and, >8.0 ng/dL, respectively), caused by a novel heterozygous frameshift mutation in exon 10 of the thyroid hormone receptor beta gene (THRB), c.1347-1357 del actcttccccc : p.E449DfsX11. RTH was detected at the neonatal screening program. At 4 years of age, the patient continued to suffer from mental retardation, hyperactivity, insomnia, and reduced resting energy expenditure (REE), despite daily thyroxine (L-T4) therapy. Every-other-day high-dose liothyronine (L-T3) therapy improved her symptoms and increased her REE, without thyrotoxicosis. CONCLUSION: In a case of severe RTH, every-other-day L-T3 administration enhanced REE and psychomotor development, without promoting symptoms of thyrotoxicosis. Every-other-day L-T3 administration may be an effective strategy for the treatment of severe RTH.

Molecular characterization of human thyroid hormone receptor ß isoform 4.

Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor ß isoform (TRß4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRß4 from a molecular basis. Temporal expression of TRß4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRß1 is potentially stable. TRß1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRß4 in a dose-dependent manner. Thus, TRß4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRß1 and TRß4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRß4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRß1. In contrast, TRß4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRß1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRß4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRßs, we found that both TRß1 and TRß4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRßs within the nucleus.

Thyroid receptor ß involvement in the effects of acute nicotine on hippocampus-dependent memory.

Cigarette smoking is common despite adverse health effects. Nicotine's effects on learning may contribute to addiction by enhancing drug-context associations. Effects of nicotine on learning could be direct or could occur by altering systems that modulate cognition. Because thyroid signaling can alter cognition and nicotine/smoking may change thyroid function, nicotine could affect learning through changes in thyroid signaling. These studies investigate the functional contributions of thyroid receptor (TR) subtypes ß and α1 to nicotine-enhanced learning and characterize the effects of acute nicotine and learning on thyroid hormone levels. We conducted a high throughput screen of transcription factor activity to identify novel targets that may contribute to the effects of nicotine on learning. Based on these results, which showed that combined nicotine and learning uniquely acted to increase TR activation, we identified TRs as potential targets of nicotine. Further analyses were conducted to determine the individual and combined effects of nicotine and learning on thyroid hormone levels, but no changes were seen. Next, to determine the role of TRß and TRα1 in the effects of nicotine on learning, mice lacking the TRß or TRα1 gene and wildtype littermates were administered acute nicotine prior to fear conditioning. Nicotine enhanced contextual fear conditioning in TRα1 knockout mice and wildtypes from both lines but TRß knockout mice did not show nicotine-enhanced learning. This finding supports involvement of TRß signaling in the effect of acute nicotine on hippocampus-dependent memory. Acute nicotine enhances learning and these effects may involve processes regulated by the transcription factor TRß.

Resistance to thyroid hormone due to mutations in the THRB gene impairs bone mass and affects calcium and phosphorus homeostasis.

CONTEXT: Resistance to thyroid hormone (RTH) is an inherited syndrome of reduced tissue responsiveness to thyroid hormone, which is usually due to mutations in the thyroid hormone receptor ß gene (THRB). Few studies have been conducted to investigate bone and mineral metabolism in RTH. OBJECTIVE: The objective of the study was to evaluate the clinical and biochemical parameters related to bone and mineral metabolism in RTH due to mutations in the THRB gene (RTHß). DESIGN AND PARTICIPANTS: We conducted a cross-sectional study on 14 patients with RTHß (RTHG), eight adults and six children, and 24 control subjects (CG). OUTCOMES: Serum measures included total calcium (TCa), inorganic phosphate (iP), alkaline phosphatase (AP), parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD), osteocalcin (OC), carboxyterminal telopeptide (CTX), and fibroblast growth factor 23 (FGF-23). We estimated the renal threshold phosphate concentration (TmPO4/GFR) and assessed bone mass using dual X-ray absorptiometry. RESULTS: Adults and children with RTH showed higher serum levels of TCa than controls (P=.029 and, P=.018 respectively). However, only children with RTH exhibited lower serum levels of iP than controls (P=.048). FGF-23 was higher in RTHß children (P=.04). RTHß adults had lower whole-body (P=.01) and lumbar spine (P=.01) bone mineral density than control subjects. The same pattern was observed when the results were expressed as Z-scores between groups, with a lower value in RTHG than in CG for the lumbar spine of adults (P=.03). No difference was observed between groups in PTH, 25OHD, AP, OC, and CTX. CONCLUSION: Biochemical abnormalities are seen in children with RTH (Low iP, high FGF23), while high calcium (with normal UCa) is seen in RTH subjects of all ages, and later on, in adult life, low BMD is seen. Considering that the TRα1 isoform is the predominant TR in the skeleton, we hypothesize that probably these patients may exhibit enhanced calcium flux from bone to circulation. Our data represent a challenge for new studies to unveil the control of calcium and phosphorus homeostasis and fracture risk in these patients.

Maternal high-fat diet modulates the fetal thyroid axis and thyroid gene expression in a nonhuman primate model.

Thyroid hormone (TH) is an essential regulator of both fetal development and energy homeostasis. Although the association between subclinical hypothyroidism and obesity has been well studied, a causal relationship has yet to be established. Using our well-characterized nonhuman primate model of excess nutrition, we sought to investigate whether maternal high-fat diet (HFD)-induced changes in TH homeostasis may underlie later in life development of metabolic disorders and obesity. Here, we show that in utero exposure to a maternal HFD is associated with alterations of the fetal thyroid axis. At the beginning of the third trimester, fetal free T(4) levels are significantly decreased with HFD exposure compared with those of control diet-exposed offspring. Furthermore, transcription of the deiodinase, iodothyronine (DIO) genes, which help maintain thyroid homeostasis, are significantly (P < 0.05) disrupted in the fetal liver, thyroid, and hypothalamus. Genes involved in TH production are decreased (TRH, TSHR, TG, TPO, and SLC5A5) in hypothalamus and thyroid gland. In experiments designed to investigate the molecular underpinnings of these observations, we observe that the TH nuclear receptors and their downstream regulators are disrupted with maternal HFD exposure. In fetal liver, the expression of TH receptor ß (THRB) is increased 1.9-fold (P = 0.012). Thorough analysis of the THRB promoter reveals a maternal diet-induced alteration in the fetal THRB histone code, alongside differential promoter occupancy of corepressors and coactivators. We speculate that maternal HFD exposure in utero may set the stage for later in life obesity through epigenomic modifications to the histone code, which modulates the fetal thyroid axis.

Changes of thyroid hormone levels and related gene expression in zebrafish on early life stage exposure to triadimefon.

In this study, zebrafish was exposed to triadimefon. Thyroid hormones levels and the expression of related genes in the hypothalamic-pituitary-thyroid (HPT) axis, including thyroid-stimulating hormone (TSH-beta), deiodinases (dio1 and dio2) and the thyroid hormone receptor (thraa and thrb) were evaluated. After triadimefon exposure, increased T4 can be explained by increased thyroid-stimulating hormone (TSH-beta). The conversion of T4 to T3 (deiodinase type I-dio1) was decreased, which reduced the T3 level. Thyroid hormone receptor beta (thrb) mRNA levels were significantly down-regulated, possibly as a response to the decreased T3 levels. The overall results indicated that triadimefon exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by triadimefon could occur at several steps in the synthesis, regulation, and action of thyroid hormones.

Tumor suppressor action of liganded thyroid hormone receptor beta by direct repression of beta-catenin gene expression.

The abundance of ß-catenin, which plays a critical role in oncogenesis, is tightly controlled by proteasomal pathways. Its aberrant accumulation is associated with the overactivation of its oncogenic signaling and tumorigenesis in cancers, including thyroid cancer. Our previous studies have suggested that ß-catenin abundance could also be regulated at the transcriptional level by thyroid hormone (T(3)) and thyroid hormone receptor ß (TRß). By using hypothyroid mice supplemented or not with T(3), we showed that T(3) significantly repressed Ctnnb1 expression in vivo in the thyroid. By using two human cell lines, i.e., the thyroid HTori and the cervical cancer HeLa cell lines, each stably expressing TRß, we observed that T(3) induced the down-regulation of CTNNB1 transcript levels. Luciferase reporter assays with various constructs harboring 5' deletion of the CTNNB1 promoter or with mutated thyroid hormone response element (TRE) binding sites, and EMSAs showed that this transrepression was mediated through an interaction between TRß-retinoid X receptor ß complexes and TREs located in the human CTNNB1 promoter between -807 and -772 and consisting of two hexamers separated by 14 nucleotides. The direct regulation of CTNNB1 expression by TRß was further confirmed by chromatin immunoprecipitation assays showing TRß recruitment to the CTNNB1 promoter in thyroid cells. This is the first report demonstrating a direct repression of the ß-catenin gene by liganded TRß through interaction with negative TREs located in CTNNB1 promoter. Importantly, this study uncovers a new molecular mechanism whereby liganded TRß acts as a tumor suppressor via inhibition of the expression of a potent tumor promoter, the CTNNB1 gene.

Thyroid hormones regulate selenoprotein expression and selenium status in mice.

Impaired expression of selenium-containing proteins leads to perturbed thyroid hormone (TH) levels, indicating the central importance of selenium for TH homeostasis. Moreover, critically ill patients with declining serum selenium develop a syndrome of low circulating TH and a central downregulation of the hypothalamus-pituitary-thyroid axis. This prompted us to test the reciprocal effect, i.e., if TH status would also regulate selenoprotein expression and selenium levels. To investigate the TH dependency of selenium metabolism, we analyzed mice expressing a mutant TH receptor α1 (TRα1+m) that confers a receptor-mediated hypothyroidism. Serum selenium was reduced in these animals, which was a direct consequence of the mutant TRα1 and not related to their metabolic alterations. Accordingly, hyperthyroidism, genetically caused by the inactivation of TRß or by oral TH treatment of adult mice, increased serum selenium levels in TRα1+m and controls, thus demonstrating a novel and specific role for TRα1 in selenium metabolism. Furthermore, TH affected the mRNA levels for several enzymes involved in selenoprotein biosynthesis as well as serum selenoprotein P concentrations and the expression of other antioxidative selenoproteins. Taken together, our results show that TH positively affects the serum selenium status and regulates the expression of several selenoproteins. This demonstrates that selenium and TH metabolism are interconnected through a feed-forward regulation, which can in part explain the rapid parallel downregulation of both systems in critical illness.

T3 differentially regulates TRH expression in developing hypothalamic neurons in vitro.

Triiodothyronine (T3) plays an important role during development of the central nervous system. T3 effects on gene expression are determined in part by the type of thyroid hormone receptors (TRs) expressed in a given cell type. Previous studies have demonstrated that thyrotropin releasing hormone (TRH) transcription in the adult hypothalamus is subjected to negative regulation by thyroid hormones. However, the role of T3 on the development of TRH expression is unknown. In this study we used primary cultures derived from 17-day-old fetal rat hypothalamus to analyze the effects of T3 on TRH gene expression during development. T3 increased TRH mRNA expression in immature cultures, but decreased it in mature cultures. In addition, T3 up-regulated TRalpha1 and TRbeta2 mRNA expression. TRalpha1 expression coincided chronologically with that of TRH in the rat hypothalamus in vivo. Maturation of TRH expression in the hypothalamus may involve T3 acting through TRalpha1.

Thyroid hormone receptor ß mediates acute illness-induced alterations in central thyroid hormone metabolism.

Acute illness in mice profoundly affects thyroid hormone metabolism in the hypothalamus and pituitary gland. It remains unknown whether the thyroid hormone receptor (TR)-ß is involved in these changes. In the present study, we investigated central thyroid hormone metabolism during lipopolysaccharide (LPS)-induced illness in TRß(-/-) mice compared to wild-type (WT) mice. We administered a sublethal dose of LPS or saline to TRß(-/-) and WT mice. TRß(-/-) mice displayed higher basal levels of serum triiodothyronine (T(3)) and thyroxine (T(4)) compared to WT, reflecting thyroid hormone resistance. In the periventricular area of the hypothalamus, we observed a marked decrease in thyrotrophin-releasing hormone (TRH) mRNA expression in TRß(-/-) and WT mice at t = 4 h, coinciding with the peak in plasma corticosterone. The decrease in TRH mRNA persisted in WT, but not in TRß(-/-) mice at t = 24 h. By contrast, the increase of type 2 deiodinase (D2) mRNA already present at 4 h after LPS remained significant at 24 h in TRß(-/-), but not in WT mice. LPS decreased pituitary thyroid-stimulating hormone ß mRNA expression in WT at 24 h but not in TRß(-/-) mice. The peak in pituitary D2 expression at t = 4 h in WT was absent in TRß(-/-) mice. The relative decrease in plasma T(3) and T(4) upon LPS treatment was similar in both strains, although, at t = 24 h, plasma T(3) tended to be restored in TRß(-/-) mice. Our results suggest that TRß is involved in suppression of the central component of the hypothalamic-pituitary-thyroid axis in acute illness.

Analysis of thyroid hormone receptor betaA mRNA expression in Xenopus laevis tadpoles as a means to detect agonism and antagonism of thyroid hormone action.

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 (TRalpha) and betaA (TRbetaA) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TRalpha gene expression whereas a marked up-regulation of TRbetaA 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 TRbetaA 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 TRbetaA 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 TRbetaA 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 TRbetaA expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TRbetaA mRNA up-regulation. Results of this study suggest that TRbetaA 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.

Dietary soy protein isolate and isoflavones modulate hepatic thyroid hormone receptors in rats.

Thyroid hormone receptors (TRs) are regulators of many genes involved in cholesterol and lipid metabolism. The purpose of this study was to examine the effect of soy protein isolate (SPI) and isoflavones on hepatic TRs in rats. In Expt. 1, Sprague-Dawley rats were fed diets containing either casein or alcohol-washed SPI with or without isoflavone supplementation (5-1250 mg/kg diet) for 70, 190, and 310 d. The offspring (F1) were fed the same diets as their parents (F0). In Expt. 2, Sprague-Dawley rats were fed diets containing casein or casein plus isoflavones (50-400 mg/kg diet) for 120 d. The mRNA and protein contents of the hepatic TRs were measured by semiquantitative RT-PCR and Western blot, respectively. TRalpha1, TRalpha2, and TRbeta2 contents were not affected by SPI. However, the content of the 52-kDa TRbeta1 protein, the major isoform present in the liver, was markedly increased by dietary SPI in both sexes of F0 and F1 compared with casein. The supplemental isoflavones had no effect on TRbeta1, whereas the high doses of isoflavones (250 and 1250 mg/kg diet) reduced the hepatic TRalpha1 protein content in F1 male rats on d 28. SPI had no effect on total T3 and T4 levels. However, higher dose of supplemental isoflavones markedly increased T4 level in female rats. Overall, this study demonstrates for the first time that SPI upregulates hepatic TRbeta1 expression, and that isoflavones reduce the hepatic TRalpha1 level in young male rats. The SPI-induced TRbeta1 may play a role in mediating the hypocholesterolemic and lipid-lowering actions of soy protein.

Both thyroid hormone receptor (TR)beta 1 and TR beta 2 isoforms contribute to the regulation of hypothalamic thyrotropin-releasing hormone.

Thyroid hormones (TH) are essential regulators of vertebrate development and metabolism. Central mechanisms governing their production have evolved, with the beta-TH receptor (TRbeta) playing a key regulatory role in the negative feedback effects of circulating TH levels on production of hypothalamic TRH and hypophyseal TSH. Both TRbeta-isoforms (TRbeta1 and TRbeta2) are expressed in the hypothalamus and pituitary. However, their respective roles in TH-dependent transcriptional regulation of TRH are undefined. We confirmed the preferential role of TRbeta vs. TRalpha isoforms in TRH regulation in wild-type mice in vivo by using the TRbeta preferential agonist GC-1. We next determined the effects of tissue-specific rescue of TRbeta1 and TRbeta2 isoforms by somatic gene transfer in hypothalami of TRbeta null (TRbeta(-/-)) mice. TH-dependent TRH transcriptional repression was impaired in TRbeta(-/-) mice, but was restored by cotransfection of either TRbeta1 or TRbeta2 into the hypothalamus. TRbeta1, but not TRbeta2, displayed a role in ligand-independent activation. In situ hybridization was used to examine endogenous TRH expression in the paraventricular nucleus of the hypothalamus of TRbeta(-/-) or TRalpha null (TRalpha(o/o)) mice under different thyroid states. In contrast to published data on TRbeta2(-/-) mice, we found that both ligand-independent TRH activation and ligand-dependent TRH repression were severely impaired in TRbeta(-/-) mice. This study thus provides functional in vivo data showing that both TRbeta1 and TRbeta2 isoforms have specific roles in regulating TRH transcription.

Molecular cloning and developmental expression patterns of thyroid hormone receptors and T3 target genes in the turbot (Scophtalmus maximus) during post-embryonic development.

Thyroid hormones (TH) are pleiotropic factors important for many developmental and physiological functions in vertebrates and particularly in amphibian metamorphosis. Their effects are mediated by two specific receptors (TRalpha and TRbeta), which are ligand-dependent transcription factors, members of the nuclear hormone receptor superfamily. Besides their pivotal role in amphibian metamorphosis, TH are also critical for fish metamorphosis. As this later role of TH is less studied, we analyzed their action in the turbot (Scophtalmus maximus), a metamorphosing flat fish. We describe the isolation of sequences for the turbot orthologs of a number of Xenopus genes, which are induced during amphibian metamorphosis. Developmental expression of these genes during turbot metamorphosis was studied by several methods and the expression patterns of these genes compared with those in Xenopus and flounder. We find that the period between the onset and the end of eye migration (day 22 to day 30 post-hatching) most likely corresponds to the metamorphic climax with either high TRalpha or high TH levels. Our results show that in contrast to amphibians, it is TRalpha and not TRbeta mRNA that is up-regulated during metamorphosis. Our results highlight the notion that TH regulates, through a rise of TR expression, a genetic cascade during turbot metamorphosis. The fact that TH regulates metamorphosis in amphibian and teleost fishes suggests that TH-regulated metamorphosis is a post-embryonic process conserved in most vertebrates.

Cardiac expression and function of thyroid hormone receptor beta and its PV mutant.

Thyroid hormone (T3) influences cardiac function, and mice with deletion of thyroid hormone receptor (TR)alpha have diminished cardiac function. TR alpha 1 represents 70% and TR beta 1 represents the remaining 30% of TR in ventricular myocytes, and its role in cardiac function is not well established. To determine the role of TR beta 1 in detail, we compared contractility in isolated perfused hearts from wild-type (WT) and TR beta knockout mice under normal and increased work load. TR beta knockout hearts showed contractile function similar to WT hearts at baseline and under conditions of enhanced demand. To gain insight into the role of TR beta, we used mice with a homozygous mutation in exon 10 of TR beta encoding the dominant negative PV mutant (TR beta PV) expressed from the endogenous TR beta promoter. TR beta PV mice treated with 6-propyl-2-thiouracil and supplemented with T3 to make them euthyroid have decreased contractility with negative and positive rates of relaxation and contraction as well as peak systolic pressure diminished by 35 +/- 5, 34 +/- 6, and 35 +/- 6% in comparison with WT mice. Heart rate is diminished by 36 +/- 7%, which is accompanied by decreased expression of the pacemaker-related gene hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4). The expression of TR beta 1 in the pacemaker myocytes of the sinoatrial node was confirmed by quantitation of TR alpha 1 and TR beta 1 mRNA in sinoatrial node, which showed that TR beta 1 mRNA represents 27.5 +/- 1.6% of the ligand-binding isoforms of the TR. In summary, although TR beta is expressed at much lower levels in all regions of the heart than TR alpha 1, expression of the strong dominant negative TR beta PV mutant results in decreased contractile function and heart rate.

Identification of the downstream targets of SIM1 and ARNT2, a pair of transcription factors essential for neuroendocrine cell differentiation.

SIM1 and ARNT2 are two basic helix-loop-helix/PAS (Per-Arnt-Sim) transcription factors that control the differentiation of neuroendocrine lineages in the mouse hypothalamus. Heterozygous Sim1 mice also develop early onset obesity, possibly due to hypodevelopment of the hypothalamus. Although SIM1 and ARNT2 form heterodimers to direct the same molecular pathway, knowledge of this pathway is limited. To facilitate the identification of their downstream genes, we combined an inducible gene expression system in a neuronal cell line with microarray analysis to screen for their transcriptional targets. This method identified 268 potential target genes of SIM1/ARNT2 that displayed >1.7-fold induced expression. 15 of these genes were subjected to Northern analysis, and a high percentage of them were confirmed to be up-regulated. In vivo, several of these genes showed neuroendocrine hypothalamic expression correlating with that of Sim1. Furthermore, we found that expression of two of these potential targets, the Jak2 and thyroid hormone receptor beta2 genes, was lost in the neuroendocrine hypothalamus of the Sim1 mutant. The expression and predicted functions of many of these genes provide new insight into both the Sim1/Arnt2 action in neuroendocrine hypothalamus development and the molecular basis for the Sim1 haplo-insufficient obesity phenotype.