Dominant-negative Thyroid Hormone Receptor Research Articles

A Versatile, Behavioral Method to Investigate Thyroid Hormone Effects on Cerebellar Function.

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early life such as congenital hypothyroidism, patients display neurological disorders such as cognitive retardation and motor deficits. There are various studies using mouse models with tissue- or cell-specific TH deficiency to investigate the role of TH in the cerebellum. Compared to generalized congenital hypothyroid mice, cerebellar cell-specific TH-deficient mice display milder and subtler ataxic features, making the assessment of motor function difficult when using conventional tests such as the rotarod test. Due to the need for an alternative tool to assess motor function in TH-related animal models, we developed a versatile behavioral method called the "ladder beam test," in which we can design the various ladder tests depending on the severity of ataxia in model mice. We utilized transgenic mice expressing a dominant-negative TH receptor specifically in the cerebellar Purkinje cell, a sole output neuron in the cerebellar cortex modulating motor performance. The newly-built ladder beam test successfully detected robust impairments in motor performance in the transgenic mice at a greater level compared to the rotarod test. Disruption of motor learning was also detected in the ladder beam test but not in the rotarod test. The protocol with this novel behavioral apparatus can be applied to other animal models that may show mild ataxic phenotype to examine subtle changes in cerebellar function.

In vitro and in vivo roles of glucocorticoid and vitamin D receptors in the control of neonatal cardiomyocyte proliferative potential

Cardiomyocyte (CM) proliferative potential varies considerably across species. While lower vertebrates and neonatal mammals retain robust capacities for CM proliferation, adult mammalian CMs lose proliferative potential due to cell-cycle withdrawal and polyploidization, failing to mount a proliferative response to regenerate lost CMs after cardiac injury. The decline of murine CM proliferative potential occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extrauterine life. We recently demonstrated that thyroid hormone (TH) signaling functions as a primary factor driving CM proliferative potential loss in vertebrates. Whether other hormonal pathways govern this process remains largely unexplored. Here we showed that agonists of glucocorticoid receptor (GR) and vitamin D receptor (VDR) suppressed neonatal CM proliferation. We next examined CM nucleation and proliferation in neonatal mutant mice lacking GR or VDR specifically in CMs, but we observed no difference between mutant and control littermates at postnatal day 14. Additionally, we generated compound mutant mice that lack GR or VDR and express dominant-negative TH receptor alpha in their CMs, and similarly observed no increase in CM proliferative potential compared to dominant-negative TH receptor alpha mice alone. Thus, although GR and VDR activation is sufficient to inhibit CM proliferation, they seem to be dispensable for neonatal CM cell-cycle exit and polyploidization in vivo. In addition, given the recent report that VDR activation in zebrafish promotes CM proliferation and tissue regeneration, our results suggest distinct roles of VDR in zebrafish and rodent CM cell-cycle regulation.

Hematopoiesis in aged female mice devoid of thyroid hormone receptors.

Hypothyroidism is often associated with anemia and immunological disorders. Similar defects are found in patients and in mice with a mutated dominant-negative thyroid hormone receptor α (TRα) and in knockout mice devoid of this receptor, suggesting that this isoform is responsible for the effects of the thyroid hormones in hematopoiesis. However, the hematological phenotype of mice lacking also TRβ has not yet been examined. We show here that TRα1/TRβ-knockout female mice, lacking all known thyroid hormone receptors with capacity to bind thyroid hormones, do not have overt anemia and in contrast with hypothyroid mice do not present reduced Gata1 or Hif1 gene expression. Similar to that found in hypothyroidism or TRα deficiency during the juvenile period, the B-cell population is reduced in the spleen and bone marrow of ageing TRα1/TRβ-knockout mice, suggesting that TRβ does not play a major role in B-cell development. However, splenic hypotrophy is more marked in hypothyroid mice than in TRα1/TRβ-knockout mice and the splenic population of T-lymphocytes is not significantly impaired in these mice in contrast with the reduction found in hypothyroidism. Our results show that the overall hematopoietic phenotype of the TRα1/TRβ-knockout mice is milder than that found in the absence of hormone. Although other mechanism/s cannot be ruled out, our results suggest that the unoccupied TRs could have a negative effect on hematopoiesis, likely secondary to repression of hematopoietic gene expression.

SUN-338 Inflammation Promotes Aggressive Thyroid Cancer Progression in a Mouse Model

Thyroid cancer is the most common endocrine malignancy. The association between inflammation and thyroid cancer has long been recognized. However, a cause-effect relationship at the molecular level has yet to be elucidated. We have created a mutant mouse, which expresses a dominant negative thyroid hormone receptor β (denoted as TRβPV) and deficiency in one allele of the Pten gene (ThrbPV/PVPten+/- mice). This mutant mouse exhibits aggressive follicular thyroid cancer similarly as in patients. We explored how inflammation could contribute to thyroid carcinogenesis in ThrbPV/PVPten+/- mice. Using FACS analysis, we found significantly increased inflammatory monocytes in thyroid tumors of ThrbPV/PVPten+/- mice. We next identified alterations in inflammation-related genes during thyroid carcinogenesis by cDNA microarrays to compare global gene expression profiles between thyroids of wild-type and ThrbPV/PVPten+/- mice. Gene array data revealed that a total 2,410 genes were differentially altered in thyroid tumors of ThrbPV/PVPten+/- mice (1,097 genes were up-regulated, and 1,313 genes were down-regulated). Analysis of the expression data showed an enrichment of genes that were associated with immune cell markers, cytokines, and chemokines, including Ptgs1, Sphk1, OPN, Tnfrsf18, CCL12, and IL6. Using Q/PCR, we validated the array data to show the significant increase in the expression of pro-inflammatory target genes [such as Ptgs1 (3 folds); Sphk1 (7.5 folds); OPN (5.4 folds), MCP1 (2.5 folds), IL17 (10.3 folds) and Tnfrsf18 (9.4 folds)] and significant decrease in the expression of anti-inflammatory genes [Ephx2 (2.3 folds) and CD163 (5.9 folds)] in thyroid tumors of ThrbPV/PVPten+/- mice. We further delineated the inflammatory role of osteopontin (OPN) in the thyroid carcinogenesis of ThrbPV/PVPten+/- mice. OPN is thought to facilitate the recruitment of monocytes/macrophages and to mediate cytokine secretion in leukocytes. Extracellular OPN functions through its interaction with multiple cell surface receptors (e.g., integrins and CD44), leading to the activation of PI3K-AKT and NF-kB signaling. We found that the protein abundance of OPN and integrin β1 was highly increased and concordantly, the downstream effectors, AKT and NF-kB were significantly elevated to drive thyroid tumor progression of ThrbPV/PVPten+/- mice. These results demonstrated that increased inflammation driven by elevated expression of immune-related genes and cytokines promoted thyroid cancer progression and that OPN played important roles in thyroid carcinogenesis. These preclinical findings suggest that OPN can be a potential target for thyroid cancer therapy.

Expression of dominant-negative thyroid hormone receptor alpha1 in Leydig and Sertoli cells demonstrates no additional defect compared with expression in Sertoli cells only.

BackgroundIn the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial.MethodsThe TRαAMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TRα1 (thyroid receptor α1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter.FindingsWe showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TRαAMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TRαAMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TRαAMI-ARO males showed normal fertility. This phenotype is similar to TRαAMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TRαAMI-ARO.Conclusions/SignificanceWe concluded that the presence of a mutant TRαAMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TRα1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis.

Aberrant cerebellar development of transgenic mice expressing dominant-negative thyroid hormone receptor in cerebellar Purkinje cells.

To study the role of the thyroid hormone (TH) in cerebellar development, we generated transgenic mice expressing a dominant-negative TH receptor (TR) in cerebellar Purkinje cells. A mutant human TRβ1 (G345R), which binds to the TH-response element but cannot bind to T3, was subcloned into exon 4 of the full-length L7/Pcp-2 gene, which is specifically expressed in Purkinje and retinal rod bipolar cells. The transgene was specifically expressed in Purkinje cells in the postnatal cerebellum. Purkinje cell dendrite arborization was significantly delayed in the transgenic mice. Surprisingly, granule cell migration was also significantly delayed. In the primary cerebellar culture, TH-induced Purkinje cell dendrite arborization was also suppressed. In quantitative real-time RT-PCR analysis, the expression levels of several TH-responsive genes were altered. The expression levels of inositol trisphosphate receptor type 1 and retinoic acid receptor-related orphan receptorα mRNAs, which are mainly expressed in Purkinje cells, and brain-derived neurotrophic factor mRNA, which is expressed in both Purkinje and granule cells, were significantly decreased. The expression levels of neurotrophin-3 and hairless mRNAs, which are mainly expressed in granule cells, and myelin basic protein mRNA, which is mainly expressed in oligodendrocytes, were also decreased. The motor coordination of transgenic mice was significantly disrupted. These results indicate that TH action through its binding to TR in Purkinje cells is required for the normal cerebellar development. TH action through TR in Purkinje cells is also important for the development of other subsets of cerebellar cells such as granule cells and oligodendrocytes.

Reactivation of the Silenced Thyroid Hormone Receptor β Gene Expression Delays Thyroid Tumor Progression

That a knock-in mouse harboring a dominant-negative thyroid hormone receptor (TR)-β (Thrb) mutation develops metastatic thyroid cancer strongly suggests the involvement of TRβ in carcinogenesis. Epigenetic silencing of the THRB gene is common in human cancers. The aim of the present study was to determine how DNA methylation affected the expression of the THRB gene in differentiated thyroid cancer (DTC) and how reexpression of the THRB gene attenuated the cancer phenotypes. We used methylation-specific PCR to examine the expression and promoter methylation of the THRB gene in DTC tissues. Thyroid cancer cells with hypermethylated THRB were treated with the demethylating agents 5'-aza-2'-deoxycytidine (5'-aza-CdR) and zebularine to evaluate their impact on the cancer cell phenotypes. THRB mRNA expression in DTC was 90% lower than in normal controls, and this decrease was associated with a higher tumor/lymph node staging. The promoter methylation level of the THRB gene had a significant negative correlation with the expression level of the THRB gene. Treatment of FTC-236 cells with 5'-aza-CdR or zebularine induced reexpression of the THRB gene and inhibited cell proliferation and migration. FTC-236 cells stably expressing TRβ exhibited lower cell proliferation and migration through inhibition of β-catenin signaling pathways compared with FTC-236 without TRβ. 5'-Aza-CdR also led to suppression of tumor growth in an in vivo xenograft model using FTC-236 cells consistent with the cell-based studies. These finding indicate that TRβ is a tumor suppressor and could be tested as a potential therapeutic target.

A Conserved Lysine in the Thyroid Hormone Receptor–α1 DNA-Binding Domain, Mutated in Hepatocellular Carcinoma, Serves as a Sensor for Transcriptional Regulation

Nuclear receptors are hormone-regulated transcription factors that play key roles in normal physiology and development; conversely, mutant nuclear receptors are associated with a wide variety of neoplastic and endocrine disorders. Typically, these receptor mutants function as dominant negatives and can interfere with wild-type receptor activity. Dominant-negative thyroid hormone receptor (TR) mutations have been identified in over 60% of the human hepatocellular carcinomas analyzed. Most of these mutant TRs are defective for corepressor release or coactivator binding in vitro, accounting for their transcriptional defects in vivo. However, two HCC-TR mutants that function as dominant-negative receptors in cells display near-normal properties in vitro, raising questions about the molecular basis behind their transcriptional defects. We report here that a single amino acid substitution, located at the same position in the DNA-binding domain of both mutants, is responsible for their impaired transcriptional activation and dominant-negative properties. Significantly, this amino acid, K74 in TRalpha, is highly conserved in all known nuclear receptors and seems to function as an allosteric sensor that regulates the transcriptional activity of these receptors in response to binding to their DNA recognition sequences. We provide evidence that these two human hepatocellular carcinoma mutants have acquired dominant-negative function as a result of disruption of this allosteric sensing. Our results suggest a novel mechanism by which nuclear receptors can acquire transcriptional defects and contribute to neoplastic disease.

Cell–cell interactions during remodeling of the intestine at metamorphosis in Xenopus laevis

Amphibian metamorphosis is accompanied by extensive intestinal remodeling. This process, mediated by thyroid hormone (TH) and its nuclear receptors, affects every cell type. Gut remodeling in Xenopus laevis involves epithelial and mesenchymal proliferation, smooth muscle thickening, neuronal aggregation, formation of intestinal folds, and shortening of its length by 75%. Transgenic tadpoles expressing a dominant negative TH receptor (TRDN) controlled by epithelial-, fibroblast-, and muscle-specific gene promoters were studied. TRDN expression in the epithelium caused abnormal development of virtually all cell types, with froglet guts displaying reduced intestinal folds, thin muscle and mesenchyme, absence of neurons, and reduced cell proliferation. TRDN expression in fibroblasts caused abnormal epithelia and mesenchyme development, and expression in muscle produced fewer enteric neurons and a reduced inter-muscular space. Gut shortening was inhibited only when TRDN was expressed in fibroblasts. Gut remodeling results from both cell-autonomous and cell–cell interactions.

Impaired Ca 2+ handling and contraction in cardiomyocytes from mice with a dominant negative thyroid hormone receptor α 1

The profound effects of thyroid hormone (TH) on heart development and function are mediated by the thyroid hormone receptors (TR) α 1 and β 1. While numerous patients with TRβ 1 mutations have been identified, patients with similar mutations in TRα 1 are yet to be discovered. Recently generated heterozygous mice with a dominant negative mutation in TRα 1 (TRα 1+/m mice) have normal TH levels, which may have hampered the discovery of patients with such mutations. We now measure intracellular Ca 2+ and contraction in cardiomyocytes isolated from TRα 1+/m mice and wildtype littermates (WT). TRα 1+/m cardiomyocytes showed a phenotype similar to that in hypothyroidism with significant slowing of voltage-activated Ca 2+ transients and contractions. Increased stimulation frequency (from 0.5 to 3 Hz) or β-adrenergic stimulation reduced the differences between TRα 1+/m and WT cardiomyocytes. However, in TRα 1+/m cells stimulation at 3 Hz gave a marked increase in diastolic Ca 2+ and β-adrenergic stimulation triggered spontaneous Ca 2+ release events during relaxation. Both TRα 1+/m and WT cardiomyocytes responded to TH treatment by displaying a “hyperthyroid” phenotype with faster and larger Ca 2+ transients and contractions. Excised TRα 1+/m hearts showed an increased expression of phospholamban (PLB). In conclusion, isolated TRα 1+/m cardiomyocytes display major dysfunctions with marked slowing of the Ca 2+ transients and contractions.

Neurological abnormalities in mice with a dominant negative thyroid hormone receptor alpha 1

Neurological abnormalities in mice with a dominant negative thyroid hormone receptor alpha 1

Dual mechanisms governing muscle cell death in tadpole tail during amphibian metamorphosis.

The tadpole tail, which is twice as long as the body, is induced to resorb completely by thyroid hormone within several days during the anuran metamorphosis. To investigate the underlying mechanism, we undertook two approaches. First, we examined the effect of dominant-negative thyroid hormone receptor (DNTR) on muscle cell death in vitro. The overexpression of DNTR suppressed the death of a tail-derived myoblastic cell line induced by thyroid hormone. Second, tadpole tails were injected with a reporter gene and the DNTR expression construct, and the reporter gene expression in muscle cells was followed during the spontaneous metamorphosis. DNTR overexpression inhibited a decrease of the reporter gene expression that began at stage 57 in the control tadpoles but only delayed massive muscle cell death at stage 63 when tails shrink very rapidly. Some remained even a few weeks after the metamorphosis, although most DNTR-overexpressing cells died by the end of the metamorphosis. These results led us to propose that thyroid hormone induces the suicide of muscle cells (the cell-autonomous death) in the tail between stage 57 and 62 and that both the murder and suicide mechanisms execute muscle cell death in stage 62-64 to remove muscle promptly and completely.

Tadpole skin dies autonomously in response to thyroid hormone at metamorphosis.

Transgenic tadpoles that express a dominant negative thyroid hormone (TH) receptor specifically in their skin undergo normal metamorphosis, with one exception: they retain a larval epidermis over the developing adult epithelium. TH-induced death of the tadpole epidermis is inhibited by the dominant negative TH receptor whereas the TH-induced response of the neighboring fibroblasts and the cells that form the adult skin occur normally. Therefore death of the tadpole skin is a direct and cell autonomous target of TH, and its protection has no detectable influence on TH-induced changes of other cell types.

Diverse developmental programs of Xenopus laevis metamorphosis are inhibited by a dominant negative thyroid hormone receptor.

Metamorphosis of anuran tadpoles is controlled by thyroid hormone (TH). Here we demonstrate that transgenic Xenopus laevis tadpoles expressing a dominant negative form of TH receptor-alpha are resistant to a wide variety of the metamorphic changes induced by TH. This result confirms that TH receptors mediate both early and late developmental programs of metamorphosis as diverse as growth in the brain, limb buds, nose and Meckel's cartilage, remodeling of the intestine, and death and resorption of the gills and tail.

Asymmetric growth and development of the Xenopus laevis retina during metamorphosis is controlled by type III deiodinase.

During the metamorphosis of the Xenopus laevis retina, thyroid hormone (TH) preferentially induces ventral ciliary marginal zone (CMZ) cells to both increase their proliferation and give rise to ipsilaterally projecting ganglion cells. Here we show that dorsal CMZ cells express type III deiodinase (D3), an enzyme that inactivates TH. The dorsal CMZ cells can be induced to proliferate if deiodinase activity is inhibited. D3 or dominant-negative thyroid hormone receptor transgenes inhibit both TH-induced proliferation of the ventral CMZ cells and the formation of the ipsilateral projection. Thus, the localized expression of D3 in the dorsal CMZ cells accounts for the asymmetric growth of the frog retina.

Unravelling of physiological functions of retinoids using a dominant-negative retinoic acid receptor

We have constructed dominant-negative retinoic acid receptors (RARs) by substituting single amino acid which has been found in a dominant-negative thyroid hormone receptor, and have expressed the dominant-negative RAR in the epidermis, a potential target organ of retinoic acid (RA). The resultant transgenic mice exhibited dramatic suppression of epidermal development, demonstrating the absolute requirement of RA in normal skin development. This method is theoretically applicable to every organ, thus opening the way to define the physiological roles of RA during embryogenesis as well as in adults.

Inhibition of skin development by targeted expression of a dominant-negative retinoic acid receptor.

Although pharmacological doses of retinoic acid (RA) have a wide variety of actions in vivo, experimental difficulties have prevented a definitive assignment of its physiological functions. We recently made a dominant-negative retinoic acid receptor (RAR) by a single amino-acid substitution which creates a dominant-negative thyroid hormone receptor. The mutated RAR efficiently inhibited the endogenous activities of RARs (alpha, beta, gamma). Thus, targeted expression of the mutated receptor should reveal RA functions during organogenesis by blocking RA signalling in the tissues concerned. To address this possibility, we expressed the dominant-negative RAR in the epidermis, a potential target organ of RA. We report here that the resultant transgenic mice exhibited dramatic suppression of epidermal maturation, demonstrating the requirement of RA in normal skin development.

T<SUB>3</SUB> Binding Properties of Nuclear Fibroblast Receptors from Patients Homozygous and Heterozygous for A Dominant Negative Mutation of C-erbAβ

This study represents the first measurements of T3 binding to nuclear receptors in fibroblasts from a homozygous child with a dominant negative thyroid hormone receptor gene. Previously demonstrated in this child and kindred was a three base pair deletion in the c-erbAβ thyroid hormone receptor gene resulting in the loss of threonine codon 332. In this study using cultured skin fibroblasts from this homozygous child and his heterozygous father there was normal binding affinity and a two-fold less binding capacity in comparison to control fibroblasts. These data are consistent with loss of β-receptor T3-binding activity, but the presence of normally functioning α-receptors in vivo.

Homozygosity for a Dominant Negative Thyroid Hormone Receptor Gene Responsible for Generalized Resistance to Thyroid Hormone*

Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA beta in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the beta-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4, 330.8 pmol/L; free T3, 82,719 fmol/L). He displayed a complex mixture of tissue-specific hyperthyroidism and hypothyroidism. He had delayed growth (height age, 1 3/12 yr at chronological age 2 9/12 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardic. The homozygous patient of kindred S is markedly different from a recently reported patient with no c-erbA beta-receptor. This difference indicates that a dominant negative form of c-erbA beta in man can inhibit at least some thyroid hormone action mediated by the c-erbA alpha-receptors.