Regulation of Gene Expression by Thyroid Hormone in Primary Astrocytes: Factors Influencing the Genomic Response.

Astrocytes mediate the action of thyroid hormone in the brain on other neural cells through the production of the active hormone triiodothyronine (T3) from its precursor thyroxine. T3 has also many effects on the astrocytes in vivo and in culture, but whether these actions are directly mediated by transcriptional regulation is not clear. In this work, we have analyzed the genomic response to T3 of cultured astrocytes isolated from the postnatal mouse cerebral cortex using RNA sequencing. Cultured astrocytes express relevant genes of thyroid hormone metabolism and action encoding type 2 deiodinase (Dio2), Mct8 transporter (Slc16a2), T3 receptors (Thra1 and Thrb), and nuclear corepressor (Ncor1) and coactivator (Ncoa1). T3 changed the expression of 668 genes (4.5% of expressed genes), of which 117 were responsive to T3 in the presence of cycloheximide. The Wnt and Notch pathways were downregulated at the posttranscriptional level. Comparison with the effect of T3 on astrocyte-enriched genes in mixed cerebrocortical cultures isolated from fetal cortex revealed that the response to T3 is influenced by the degree of astrocyte maturation and that, in agreement with its physiological effects, T3 promotes the transition between the fetal and adult patterns of gene expression.

Cigarette Smoke Extract Disrupts Transcriptional Activities Mediated by Thyroid Hormones and Its Receptors.

Cigarette smoke contains over 4800 compounds, including at least 200 toxicants or endocrine disruptors. Currently, effects of cigarette smoke on thyroid hormone (TH) levels remains to be clarified. Here, we demonstrate that cigarette smoke extract (CSE) possesses thyroid hormone properties and acts synergistically as a partial agonist for thyroid hormone receptors (TRs) in the presence of TH. In transient gene expression experiments, CSE stimulated transcriptional activity with TH in a dose-dependent manner. Stimulatory effects were observed with physiological TH concentrations, although CSE did not activate TRs without TH. CSE (5%) dissolved in phosphate-buffered saline (PBS) supplemented with 1 nM TH was approximately comparable to 3.2±0.1 and 2.3±0.2 nM of TRα1 and TRß1, respectively. To illustrate probable mechanisms of the CSE agonistic activity, effects on TR mediated transcriptional functions with cofactors were investigated. With a mammalian two-hybrid assay, CSE recruited the nuclear coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC1) to the TR. Unsaturated carbonyl compounds, acrolein, crotonaldehyde, and methyl vinyl ketone, representative constituents of CSE, retained such agonistic properties and possibly contributed to stimulatory effects. The results suggest that CSE recruits a transcriptional activator and may reinforce TH binding to the TR additively, resulting in gene expression. CSE partially agonizes TH action and may disturb the function of various nuclear hormone receptor types and their cofactors to disrupt the physiological processes.

Editor's Highlight: Structure-Based Investigation on the Binding and Activation of Typical Pesticides With Thyroid Receptor.

A broad range of pesticides have been reported to interfere with the normal function of the thyroid endocrine system. However, the precise mechanism(s) of action has not yet been thoroughly elucidated. In this study, 21 pesticides were assessed for their binding interactions and the potential to disrupt thyroid homeostasis. In the GH3 luciferase reporter gene assays, 5 of the pesticides tested had agonistic effects in the order of procymidone > imidacloprid > mancozeb > fluroxypyr > atrazine. 11 pesticides inhibited luciferase activity of T3 to varying degrees, demonstrating their antagonistic activity. And there are 4 pesticides showed mixed effects when treated with different concentrations. Surface plasmon resonance (SPR) biosensor technique was used to directly measure the binding interactions of these pesticides to the human thyroid hormone receptor (hTR). 13 pesticides were observed to bind directly with TR, with a KD ranging from 4.80E-08 M to 9.44E-07 M. The association and disassociation of the hTR/pesticide complex revealed 2 distinctive binding modes between the agonists and antagonists. At the same time, a different binding mode was displayed by the pesticides showed mix agonist and antagonist activity. In addition, the molecular docking simulation analyses indicated that the interaction energy calculated by CDOCKER for the agonists and antagonists correlated well with the KD values measured by the surface plasmon resonance assay. These results help to explain the differences of the TR activities of these tested pesticides.

The expression of thyroid hormone receptors (THR) is regulated by the progesterone receptor system in first trimester placental tissue and in BeWo cells in vitro.

BACKGROUND: Thyroid hormones are essential for the maintenance of pregnancy and a deficiency in maternal thyroid hormones has been associated with early pregnancy losses. The aim of this study was a systematic investigation of the influence of mifepristone (RU 486) on the expression of the thyroid hormone receptor (THR) isoforms THRα1, THRα2, THRß1 and THRß2 on protein and mRNA-level. METHODS: Samples of placental tissue were obtained from patients with mifepristone induced termination of pregnancy (n=13) or mechanical induced termination of normal pregnancy (n=20), each from the 4th to 13th week of pregnancy. Expression of THRα1, THRα2, THRß1 and THRß2 was analysed on protein level by immunohistochemistry and on mRNA level by real time RT-PCR (TaqMan). The influence of progesterone on THR gene expression was analysed in the trophoblast tumour cell line BeWo by real time RT-PCR (TaqMan). RESULTS: Nuclear expression of THRα1, THRα2 and THRß1 is downregulated on protein level in mifepristone (RU 486) treated villous trophoblast tissue. In decidual tissue, we found a significant downregulation only for THRα1 in mifepristone treated tissue. On mRNA level, we also found a significantly reduced expression of THRA but no significant downregulation for THRB in placental tissue. The gene THRA encodes the isoform THRα and the gene THRB encodes the isoform THRß. The majority of cells expressing the thyroid hormone receptors in the decidua are decidual stromal cells. In addition, in vitro experiments with trophoblast tumour cells showed that progesterone significantly induced THRA but not THRB expression. CONCLUSIONS: Termination of pregnancy with mifepristone (RU 486) leads to a downregulation of THRα1, THRα2 and THRß1 in villous trophoblasts and in addition to a decreased expression of THRA in placental tissue. Decreased expression of THRα1 induced by RU486 could also be found in the decidua. Therefore inhibition of the progesterone receptor may be responsible for this downregulation. This assumption is supported by the finding, that stimulation of the progesterone receptor by progesterone itself up-regulated THRA in trophoblast cells in vitro.

Toxicogenomic analysis of the ability of brominated flame retardants TBBPA and BDE-209 to disrupt thyroid hormone signaling in neural cells.

Brominated flame retardants are suspected to act as disruptors of thyroid hormone signaling. This raises the concern that they might affect children's cognitive functions by influencing thyroid hormone signaling in the developing brain. We present here an in vitro analysis of the ability of the most common compounds, tetrabromobisphenol A (TBBPA) and BDE-209, to alter thyroid hormone response based on a model neural cell line and genome-wide analysis of gene expression.

Effect on metabolic enzymes and thyroid receptors induced by BDE-47 by activation the pregnane X receptor in HepG2, a human hepatoma cell line.

2,2',4,4'-Tetra-bromodiphenyl ether (BDE-47), an important congener among polybrominated diphenyl ether (PBDE) compounds, has been predominantly in environmental samples and human tissue. Thyroid disruption is the most sensitive endpoint effect among a number of health effects of exposure to BDE-47 in animals and humans. However, the detailed underlying mechanisms in humans are not well understood. In the present study, human pregnane X receptor (hPXR)-overexpressing HepG2 cell model and a dual-luciferase reporter assay system were constructed to investigate the role of hPXR in BDE-47-induced alterations of expression of metabolic enzymes and TR in vitro. The results showed that hPXR was significantly activated by BDE-47, and expression levels of both mRNA and protein of the thyroid receptor (TR) isoforms TRα1 and TRß1 were decreased in hPXR-overexpressing HepG2 cells after BDE-47 treatment. However, the increased expression of hepatic microsomal phase I enzyme CYP3A4 and phase II enzymes, UGT1A3 and SULT2A1 were also found. Taken together, the results indicated that BDE-47 was a strong hPXR activator, activation of hPXR played an important role in BDE-47-induced down-regulation of TR, and up-regulations of CYP3A4, UGT1A3, and SULT2A1 participated in the process, which may provide more toxicological evidence on mechanisms of disruption of thyroid hormone induced by BDE-47.

Receptors for thyrotropin-releasing hormone, thyroid-stimulating hormone, and thyroid hormones in the macaque uterus: effects of long-term sex hormone treatment.

OBJECTIVE: Thyroid gland dysfunction is associated with menstrual cycle disturbances, infertility, and increased risk of miscarriage, but the mechanisms are poorly understood. However, little is known about the regulation of these receptors in the uterus. The aim of this study was to determine the effects of long-term treatment with steroid hormones on the expression, distribution, and regulation of the receptors for thyrotropin-releasing hormone (TRHR) and thyroid-stimulating hormone (TSHR), thyroid hormone receptor α1/α2 (THRα1/α2), and THRß1 in the uterus of surgically menopausal monkeys. METHODS: Eighty-eight cynomolgus macaques were ovariectomized and treated orally with conjugated equine estrogens (CEE; n = 20), a combination of CEE and medroxyprogesterone acetate (MPA; n = 20), or tibolone (n = 28) for 2 years. The control group (OvxC; n = 20) received no treatment. Immunohistochemistry was used to evaluate the protein expression and distribution of the receptors in luminal epithelium, glands, stroma, and myometrium of the uterus. RESULTS: Immunostaining of TRHR, TSHR, and THRs was detected in all uterine compartments. Epithelial immunostaining of TRHR was down-regulated in the CEE + MPA group, whereas in stroma, both TRHR and TSHR were increased by CEE + MPA treatment as compared with OvxC. TRHR immunoreactivity was up-regulated, but THRα and THRß were down-regulated, in the myometrium of the CEE and CEE + MPA groups. The thyroid-stimulating hormone level was higher in the CEE and tibolone groups as compared with OvxC, but the level of free thyroxin did not differ between groups. CONCLUSIONS: All receptors involved in thyroid hormone function are expressed in monkey uterus, and they are all regulated by long-term steroid hormone treatment. These findings suggest that there is a possibility of direct actions of thyroid hormones, thyroid-stimulating hormone and thyrotropin-releasing hormone on uterine function.

Parallel biotransformation of tetrabromobisphenol A in Xenopus laevis and mammals: Xenopus as a model for endocrine perturbation studies.

The flame retardant tetrabromobisphenol A (TBBPA) is a high production flame retardant that interferes with thyroid hormone (TH) signaling. Despite its rapid metabolism in mammals, TBBPA is found in significant amounts in different tissues. Such findings highlight first a need to better understand the effects of TBBPA and its metabolites and second the need to develop models to address these questions experimentally. We used Xenopus laevis tadpoles to follow radiolabeled (14)C-TBBPA uptake and metabolism. Extensive and rapid uptake of radioactivity was observed, tadpoles metabolizing > 94% of (14)C-TBBPA within 8 h. Four metabolites were identified in water and tadpole extracts: TBBPA-glucuronide, TBBPA-glucuronide-sulfate, TBBPA-sulfate, and TBBPA-disulfate. These metabolites are identical to the TBBPA conjugates characterized in mammals, including humans. Most radioactivity (> 75%) was associated with sulfated conjugates. The antithyroid effects of TBBPA and the metabolites were compared using two in vivo measures: tadpole morphology and an in vivo tadpole TH reporter gene assay. Only TBBPA, and not the sulfated metabolites, disrupted thyroid signaling. Moreover, TBBPA treatment did not affect expression of phase II enzymes involved in TH metabolism, suggesting that the antithyroid effects of TBBPA are not due to indirect effects on TH metabolism. Finally, we show that only the parent TBBPA inhibits T3-induced transactivation in cells expressing human, zebrafish, or X. laevis TH receptor, TRα. We conclude, first, that perturbation of thyroid signaling by TBBPA is likely due to rapid direct action of the parent compound, and second, that Xenopus is an excellent vertebrate model for biotransformation studies, displaying homologous pathways to mammals.

Identical gene regulation patterns of T3 and selective thyroid hormone receptor modulator GC-1.

Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRM) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver, and insulin resistance in preclinical animal models. STRM differ from native TH in preferential binding to the TRß subtype vs. TRα, increased uptake into liver, and reduced uptake into other tissues. However, selective modulators of other nuclear receptors exhibit important gene-selective actions, which are attributed to differential effects on receptor conformation and dynamics and can have profound influences in animals and humans. Although there are suggestions that STRM may exhibit such gene-specific actions, the extent to which they are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, T(3), and the prototype STRM GC-1 induce identical gene sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TRß, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency vs. T(3), at angiopoietin-like factor 4 in HepG2. Investigation of the latter effect confirms that GC-1 acts through TRß to directly induce this gene but this gene-selective activity is not related to unusual T(3)-response element sequence, unlike previously documented promoter-selective STRM actions. Our data suggest that T(3) and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRM will be subtle and rare.

BDE-99 deregulates BDNF, Bcl-2 and the mRNA expression of thyroid receptor isoforms in rat cerebellar granular neurons.

Although the disruption of thyroid hormone (TH) signaling can largely explain the neurotoxic effects of polybrominated diphenyl ethers (PBDEs), there are still many unknowns about how this interference occurs. In this study, we expose a primary culture of rat cerebellar granule neurons (CGNs) to a 25µM concentration of one of the most prevalent PBDE congeners in humans, 2,2',4,4',5-pentaBDE (BDE-99). The main goal was to investigate the time course of BDE-99 toxicity in relation to the disruption of thyroid receptor (TR) function over 24h. In a first stage, we found that BDE-99 directly down-regulated the transcription of the isoforms TR-alpha1 and TR-alpha2, which may be a consequence of a hypothetical state that mimics hyperthyroidism. In a later stage, BDE-99 disrupted the expression of triiodothyronine (T3)-responsive genes, possibly as an effect of its metabolism. A down-regulation of the expression of the T3-mediated neurotrophin brain-derived neurotrophic factor (BDNF) and the anti-apoptotic Bcl-2 protein was also observed. Down-regulation of these two proteins was correlated with an increase in the production of reactive oxygen species (ROS). It was also found that expression of the TR-beta1 isoform, which is normally transcriptionally repressed by T3 in CGNs, was up-regulated. This up-regulation could compensate the down-regulation of the TR-alpha1 isoform, and thus slow down cell death. The dually disruptive action of BDE-99 might provide a better understanding of the potentially neurotoxic mechanism of PBDEs.

Effect of triiodothyronine on antidepressant screening tests in mice and on presynaptic 5-HT1A receptors: mediation by thyroid hormone α receptors.

Although triiodothyronine (T3) is widely used clinically, preclinical support for its antidepressant-like effects is limited, and the mechanisms are unknown. We evaluated 1) the antidepressant-like effects of T3 in the novelty suppressed feeding test (NSFT), tail suspension test (TST), and forced swim test (FST), 2) the role of presynaptic 5-HT(1A) receptors in the antidepressant-like mechanism of T3 by the hypothermic response to the 5-HT(1A) receptor agonist, 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT), 3) the thyroid hormone receptor type mediating the antidepressant-like effects by concurrent administration of the specific thyroid hormone α receptor (TRα) antagonist, dronedarone, and 4) the presence of these effects in both genders. Male and female BALB/c mice were administered 1) T3 (20, 50, 200, or 500 µg/kg per day) or vehicle or 2) T3 (50 µg/kg per day), dronedarone (100 µM/day), or the combination intraperitoneally for 21 days and then underwent a behavioral test battery. The NSFT showed a shortened latency to feed in males at the two lower T3 doses. The TST and FST showed decreased immobility in male mice at T3 doses >20 µg/kg per day and in females at all T3 doses. Concurrent dronedarone prevented T3 effects in males on the NSFT and in the TST and FST in both genders. Attenuation of 8-OH-DPAT-induced hypothermia was observed in males only and may be reduced by concurrent dronedarone. These findings support an antidepressant-like effect of T3. Attenuation of 8-OH-DPAT-induced hypothermia in males only suggests the need to evaluate a possible gender disparity in the role of presynaptic 5-HT(1A) receptors in T3 antidepressant mechanisms. Blockade by dronedarone of the antidepressant-like effects of T3 suggests that these effects are TRα receptor-mediated.

Detection of agonistic activities against five human nuclear receptors in river environments of Japan using a yeast two-hybrid assay.

A total of 16 water samples from four rivers in Japan were examined for their agonistic activities against five human nuclear receptors (estrogen receptor [ER] alpha, thyroid hormone receptor alpha, retinoic acid receptor [RAR] alpha, retinoid X receptor alpha, and vitamin D receptor) by using a yeast two-hybrid assay. The results suggest that the river environment is contaminated with endocrine disrupting chemicals (EDCs) that can interact with a variety of nuclear receptors and that contamination with those that have RAR agonistic activity may be more serious than contamination with well-known EDCs that act as ER agonists.

Detection of PBDE effects on mRNA expression in chicken (Gallus domesticus) neuronal cells using real-time RT-PCR and a new differential display method.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in a wide range of consumer products. Previous studies have suggested that PBDEs can disrupt thyroid hormone homeostasis and the developing central nervous system in rodents, but few studies have determined whether PBDEs cause similar effects in birds. An in vitro method was used to determine effects of a commercial PBDE flame retardant (DE-71) on mRNA expression in primary chicken neuronal cells derived from the cerebral hemisphere. Real-time RT-PCR assays were developed to quantify changes in mRNA abundance of genes associated with the thyroid hormone pathway; thyroid hormone receptors (TRalpha and TRbeta) and transthyretin (TTR). We also used a new differential display PCR methodology, fluorescent RNA arbitrarily primed PCR (FRAP-PCR), to determine additional effects of DE-71 on mRNA expression. Neither of the TRs responded to DE-71 exposure, but TTR mRNA decreased approximately 2-fold following exposure to 0.1, 1 and 3 microM DE-71. Candidate transcripts associated with signal transduction, neurosteroidogenesis, and neurite and axonal growth were up-regulated by DE-71 exposure. Taken together, the findings from this study indicate that this in vitro cell culture method can be used to characterize the effects of PBDEs in the avian brain.

2D QSAR studies on thyroid hormone receptor ligands.

2D QSAR studies were carried out for a series of 55 ligands for the Thyroid receptors, TRalpha and TRbeta. Significant cross-validated correlation coefficients (q(2)=0.781 (TRalpha) and 0.693 (TRbeta)) were obtained. The models' predictive abilities were proved more valuable than the classical 2D-QSAR, and were further investigated by means of an external test set of 13 compounds. The predicted values are in good agreement with experimental values, suggesting that the models could be useful in the design of novel, more potent TR ligands. Contribution map analysis identified a number of positions that are promising for the development of receptor isoform specific ligands.

Thyroid receptor ligands. Part 4: 4'-amido bioisosteric ligands selective for the thyroid hormone receptor beta.

Based on the examination of the X-ray crystallographic structures of the LBD of TRalpha and TRbeta in complex with KB-141 (2), a number of novel 4'-hydroxy bioisosteric thyromimetics were prepared. Optimal affinity and beta-selectivity (33 times), was found with a medium-sized alkyl-substituted amido group; iso-butyl (12c). It can be concluded that bioisosteric replacements of the 4'-hydroxy position represent a new promising class of TRbeta-selective synthetic thyromimetics.

Regulation of fibronectin by thyroid hormone receptors.

Thyroid hormones regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors and associated pathways. We investigated the triiodothyronine (T3) modulation of gene expression, in human hepatocellular carcinoma cell lines, via a PCR-based cDNA subtraction method. Here we present further data on one of the T3-upregulated genes, fibronectin (FN). We demonstrate that the induction of FN protein expression by T3 in TRalpha1 and TRbeta1 over-expressing cells was time and dose-dependent at the mRNA and protein levels. Blockade of protein synthesis by cycloheximide almost completely inhibited the concomitant induction of FN mRNA by T3, indicating that T3 indirectly regulates FN. Furthermore, nuclear-run on and FN promoter assay clearly can specifically increase the number of FN transcriptional demonstrated that the presence of T3 initiations. In addition, we further confirmed that the up-regulation of FN by T3 was mediated, at least in part, by transforming growth factor-beta (TGF-beta), because the induction of FN was blocked in a dose-dependent manner by the addition of TGF-beta neutralizing antibody. In an effort to elucidate the we demonstrated the involvement of the signaling pathways involved in the activation of FN by T3, mitogen activated protein kinase/c-Jun N-terminal kinase/p38 MAPK (MAPK/JNK/p38) pathway. Although T3 induces the expression of TGF-beta, neither wild-type nor dominant-negative Smad3 or Smad4 over-expression affected the activation of FN by T3. Thus, we demonstrate that T3 regulates FN gene expression indirectly at the transcriptional level, with the participation of the MAPK/JNK/p38 pathway and the TGF-beta signaling pathway but independent of Smad3/4.

Selective activation of thyroid hormone signaling pathways by GC-1: a new approach to controlling cholesterol and body weight.

The current report describes progress in development of a selective thyroid hormone receptor modulator, GC-1. This compound binds selectively to the beta-isoform of the thyroid hormone receptor, and its uptake into the heart is relatively low. Studies in rats, mice and monkeys show that GC-1 lowers cholesterol with 600- to 1400-fold more potency and approximately two- to threefold more efficacy than atorvastatin, a compound that blocks HMG-CoA reductase. GC-1 also decreases plasma levels of triglyceride and lipoprotein (a), and induces loss of fat. These effects can be observed under conditions where there is either no or minimal effect on heart rate, and no detectable loss of muscle. Although more study is required, compounds of this class deserve further investigation for treating lipid disorders and obesity.