Thyroid Hormone Receptor Signaling Research Articles

Maternal cadmium exposure impairs placental angiogenesis in preeclampsia through disturbing thyroid hormone receptor signaling.

Cadmium is a ubiquitous environmental pollutant, which can increase the risk of preeclampsia. This study was designed to determine the mechanism of cadmium exposure during pregnancy impaired placental angiogenesis that was associated with the occurrence of preeclampsia. The effects of cadmium exposure on placental thyroid hormone receptor signaling were explored. JEG3 cells were treated with CdCl2 (20μM) and the Dio2 inhibitor, IOP (100μM). Cadmium levels in maternal blood and placentae were increased in preeclampsia group. Placental angiogenesis of preeclampsia was decreased with decreased expression of PLGF and VEGF and increased expression of sFlt1. Meanwhile, the expression and nuclear translocation of thyroid hormone receptor α were decreased in preeclampsia placenta, as well as the expression of Dio2, but not the expression and nuclear translocation of thyroid hormone receptor β. Furthermore, we found that cadmium exposure downregulated the expression of thyroid hormone receptor α and Dio2, but not the expression of thyroid hormone receptor β in JEG3 cells. Also, we found that cadmium exposure decreased the expression of PLGF and VEGF and increased the expression of sFlt1 in JEG3 cells. IOP pretreatment decreased the expression of PLGF and increased the expression of sFlt1. In conclusion, our results elucidated that cadmium exposure would impair placental angiogenesis in preeclampsia through disturbing thyroid hormone receptor signaling.

Abstract LB023: Thyroid hormone receptor signaling promotes antiandrogen resistance and bone metastasis in prostate cancer

Abstract Background: Systemic factors from the tumor microenvironment (TME) promote prostate cancer (PCa) progression, metastasis, and drug resistance. Therefore, the identification of the systemic factors that can modulate the tumor cells and promote metastases will be critical to improve the efficacy of clinical treatment. Both mechanistic and functional studies revealed that acquisition of an open chromatin state on master regulators or cis-regulatory elements allow them to respond readily to microenvironment signals, thereby increasing cancer cell plasticity, tumorigenicity and malignant potential. Accordingly, analysis of open chromatin regions can help prioritize and identify risk of functionally noncoding variants. Given the power of open chromatin profiling on environmental risk prediction, we hypothesized that analysis of open chromatin regions may enable us to recognize functional trans-regulatory elements with their relevant systemic factors. Methods and Results: In human prostate cancer cells, AR blockage by enzalutamide can induces cell reprogramming, as well as transcriptional dysregulation of the heterochromatin compaction pathway. Hence, we proposed that the cells were undergoing chromatin decondensation followed by epigenetic reprogramming. To detect the genome-wide open chromatin changes in this process, we performed time course ATAC-seq and H3K27ac ChIP-seq experiments on LNCaP cells treated with enzalutamide over a 2 weeks period. Bioinformatic analysis was performed to identify the “open” enhancers and the transcriptional factors potentially bound to these enhancers. Integrated transcription factor binding motif analysis of metastatic castration-resistant prostate cancer (mCRPC) associated enhancer regions (GSE130408) and multiplexed siRNA screening revealed Thyroid Hormone Receptor Beta (THRB) as the top candidate. THRB is regulated by the thyroid hormone (T3), a systemic nuclear hormone that, in enzalutamide pretreated LNCaP, VCaP and 3PDXs, is able to induce subsequent reprogramming to a fully enzalutamide-resistant and metastatic state. Interestingly, we observed that THRB regulated genes are upregulated in T3-induced resistant prostate cancer cells. Consistently, the THRB protein, TRβ, is abundant in several aggressive PCa cell lines. Notably, knockdown of THRB reverted the growth and the bone metastatic potential of highly aggressive PCa models, such as Ptenpc-/-Smad4pc-/- and PC3M cells. Furthermore, both transcriptomic and functional analyses identified RUNX2 as a transcriptional target of THRB. In summary, we identify a novel mechanism of THRB-mediated tumor progression and drug resistance in PCa cells. Conclusion: Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. Accordingly, our results suggest a new mechanism for thyroid hormone signaling regulating metastasis potential and drug resistance. This study will provide a new perspective to identify the signals from the TME that may increase the risk of PCa progression. Citation Format: Yan Wang, Gurrapu Harsha, Dhiraj Kumar, Josue Curto, Xiaobo Wang, Filippo Giancotti. Thyroid hormone receptor signaling promotes antiandrogen resistance and bone metastasis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB023.

Acute nitrite exposure interferes with intestinal thyroid hormone homeostasis in grass carp (Ctenopharyngodon idellus)

Nitrite in the aquatic environment potentially disturbs thyroid hormone (TH) homeostasis in peripheral tissues, but little is known about TH metabolism in the intestine. This study investigated the serum concentrations of THs and thyroid-stimulating hormone (TSH) as well as the activity of intestinal iodothyronine deiodinases (IDs) of grass carp (Ctenopharyngodon idellus) exposed to various concentrations of nitrite (0, 8, 25, or 50 mg/L) for 96 h. Acute nitrite exposure significantly altered the triiodothyronine (T3) levels and the morphology of thyroid follicles at 96 h. Thyroxine (T4), free T4 levels and intestinal IDs activities showed an increase trend under nitrite stress. After 96 h exposure, nitrite down-regulated the expressions levels of intestinal Akt1 protein, sugar transporter genes, and thyroid hormone receptor (TR) signaling pathway genes except for tr ɑ1 and tr ɑ2. Moreover, the expressions levels of pparγ, cpt1α, cd36, fabp2 and fatp4 were down-regulated, whereas fabp6 and lpl were up-regulated in the 50 mg/L exposure group at 96 h. The results indicate that acute nitrite exposure has the potential to disturb the homeostasis of intestinal TH metabolism, which in turn alters TRs genes transcription, down-regulates sugar transporter activities, and promotes the energy expenditure in gut of grass carp.

Nuclear Receptor Corepressors NCoR1 and SMRT Plays Unique Roles in Central Nervous System

The nuclear corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) are critical coregulators of the thyroid hormone receptor (TR), mediating transcriptional repression via histone deacetylation. Thyroid hormone (TH) plays an essential role in many physiological processes via the TR. How the corepressors regulate TR signaling is not fully understood, especially in central nervous system (CNS). To determine the role of NCoR1 and SMRT in the CNS, we used mice with conditional NCoR1 (NCoR1lox/lox) and SMRT (SMRTlox/lox) alleles in combination with mice that express Cre recombinase in a neuronal specific fashion (Snap25-Cre). Global deletion of NCoR1 or SMRT during embryogenesis results in lethality. We also showed that NCoR1/SMRT double knock-out mice die within two weeks after induction of Cre activity in adult mice. Now, we found that neuronal specific NCoR1 or SMRT KO mice survive without obvious impairment of neuronal development. However, NCoR1/SMRT double knock-out mice die within postnatal 1-2 weeks and have impaired body growth. Thus, both NCoR1 and SMRT have important roles in maintaining normal neuronal function. Recently, cased of mutations in NCoR1 and SMRT in humans have been reported. These cases report phenotypes including Autism Spectrum Disorder (ASD) and intellectual disability. The cerebellum has been thought to contribute to motor control and learning. Surprisingly, it has also been shown to be a key brain structure involved in social cognition and its dysfunction may play a role in ASD. The Purkinje cell is the main neuron in the cerebellum. Thus, we generated cerebellar Purkinje cell specific NCoR1/SMRT knock-out mice using L7/Pcp2-Cre mice. In contrast to neuronal specific KO mice, both NCoR1 or SMRT single or double knock-out mice survive until adulthood. SMRT Purkinje cell knock-out mice showed abnormalities in 3ch social interaction test indicating impaired social functioning, similar to some ASD symptoms. Electrophysiological testing showed current injection evoked more action potentials in SMRT KO mice. These results suggest Purkinje cell dysfunction caused by SMRT deletion may result in social disability. Our data demonstrate for the first time that NCoR1 and SMRT have separate functions in different areas of the brain but also have some redundant function when knocked out together in all neurons.

Best Achievements in Translational and Basic Thyroidology in 2020.

This review discusses articles published in 2020 that presented noteworthy achievements in translational and basic thyroidology. Previously unresolved questions about thyroid hormone receptor actions and signaling mechanisms were answered using various novel in vitro and in vivo models. Using high resolution cryo-electron microscopy, the fine functional structure of thyroglobulin was demonstrated, and new insights into the pathogenesis of thyroid disease were achieved, with a focus on research into thyroid-disrupting chemicals and the gut microbiome. Novel therapeutic approaches were tried in the field of advanced thyroid cancer treatments.

Recent advances in understanding endocrine disruptors: DDT and related compounds.

Endocrine-disrupting compounds (EDCs) are environmental contaminants that modulate estrogen, androgen, and thyroid hormone receptor signaling and it has been hypothesized that human exposures to EDCs induce multiple adverse health effects. Some of these responses include male and female reproductive tract problems, obesity, and neurological/neurobehavior deficits. Extensive laboratory animal and some human studies support the EDC hypothesis. However, there is a debate among scientists and regulators regarding the adverse human health impacts of EDCs and this review highlights and gives examples of some of the concerns.

Thyroid hormone receptor α in skeletal muscle is essential for T3-mediated increase in energy expenditure.

Thyroid hormones are important for homeostatic control of energy metabolism and body temperature. Although skeletal muscle is considered a key site for thyroid action, the contribution of thyroid hormone receptor signaling in muscle to whole‐body energy metabolism and body temperature has not been resolved. Here, we show that T3‐induced increase in energy expenditure requires thyroid hormone receptor alpha 1 (TRα1) in skeletal muscle, but that T3‐mediated elevation in body temperature is achieved in the absence of muscle‐TRα1. In slow‐twitch soleus muscle, loss‐of‐function of TRα1 (TRαHSACre) alters the fiber‐type composition toward a more oxidative phenotype. The change in fiber‐type composition, however, does not influence the running capacity or motivation to run. RNA‐sequencing of soleus muscle from WT mice and TRαHSACre mice revealed differentiated transcriptional regulation of genes associated with muscle thermogenesis, such as sarcolipin and UCP3, providing molecular clues pertaining to the mechanistic underpinnings of TRα1‐linked control of whole‐body metabolic rate. Together, this work establishes a fundamental role for skeletal muscle in T3‐stimulated increase in whole‐body energy expenditure.

Pubertal fenvalerate exposure impairs cognitive and behavioral development partially through down-regulating hippocampal thyroid hormone receptor signaling

Fenvalerate, a synthetic pyrethroid insecticide, is an environmental endocrine disruptor and neurodevelopmental toxicant. An early report found that pubertal exposure to high-dose fenvalerate impaired cognitive and behavioral development. Here, we aimed to further investigate the effect of pubertal exposure to low-dose fenvalerate on cognitive and behavioral development. Mice were orally administered with fenvalerate (0.2, 1.0 and 5.0 mg/kg) daily from postnatal day (PND) 28 to PND56. Learning and memory were assessed by Morris water maze. Anxiety-related activities were detected by open-field and elevated plus-maze. Increased anxiety activities were observed only in females exposed to fenvalerate. Spatial learning and memory were damaged only in females exposed to fenvalerate. Histopathology observed numerous scattered shrinking neurons and nuclear pyknosis in hippocampal CA1 region. Neuronal density was reduced in hippocampal CA1 region of fenvalerate-exposed mice. Mechanistically, hippocampal thyroid hormone receptor (TR)β1 was down-regulated in a dose-dependent manner in females. In addition, TRα1 was declined only in females exposed to 5.0 mg/kg fenvalerate. Taken together, these suggests that pubertal exposure to low-dose fenvalerate impairs cognitive and behavioral development in a gender-dependent manner. Hippocampal TR signaling may be, at least partially, involved in fenvalerate-induced impairment of cognitive and behavioral development.

Gestational di-(2-ethylhexyl) phthalate exposure causes fetal intrauterine growth restriction through disturbing placental thyroid hormone receptor signaling

Previous study reported that gestational Di-(2-ethylhexyl) phthalate (DEHP) exposure caused fetal intrauterine growth restriction (IUGR). We aimed to investigate the role of placental thyroid hormone receptor (THR) signaling in DEHP-induced IUGR. Dams were treated with DEHP (50 or 200 mg/kg) by gavage daily throughout pregnancy. As expected, gestational DEHP exposure dose-dependently caused fetal IUGR. The mRNA levels of placental Thrα1 and Thrβ1 were reduced and nuclear translocation of placental THRα1 and THRβ1 were suppressed in DEHP-exposed mice even though thyroid hormones in maternal and fetal sera were unaffected. Correspondingly, Vegf, Pgf, Igf1 and Igf2, several THR downstream genes essential for placental angiogenesis, were down-regulated in placenta of DEHP-exposed mice. Histopathology showed that vascular space in the labyrinthine region was shrunken in placenta of DEHP-treated mice. The microvessel density in labyrinthine region was reduced in DEHP-treated mice. A nested case-control study based on MABC suggested that microvessel density was decreased in placenta of SGA cases. Moreover, protein abundance of placental THRα1 and THRβ1 were lower in SGA cases. In conclusion, gestational DEHP exposure increases fetal IUGR incidence through disturbing placental THR signaling. The present study, at least partially, elucidate the underlying mechanism of DEHP-induced fetal IUGR.

Dioxin Exposure Alters Molecular and Morphological Responses to Thyroid Hormone in Xenopus laevis Cultured Cells and Prometamorphic Tadpoles.

Amphibian metamorphosis is driven by thyroid hormone (TH). We used prometamorphic tadpoles and a cell line of the African clawed frog (Xenopus laevis) to examine immediate effects of dioxin exposure on TH. Gene expression patterns suggest cross-talk between the thyroid hormone receptor (TR) and aryl hydrocarbon receptor (AHR) signaling pathways. In XLK-WG cells, expression of Cytochrome P450 1A6 (cyp1A6), an AHR target, was induced 1000-fold by 100 nM TCDD (2, 3, 7, 8 tetrachlorodibenzo-p-dioxin). Krüppel-Like Factor 9 (klf9), the first gene induced in a cascade of TH responses tied to metamorphosis, was upregulated over 5-fold by 50 nM triiodothyronine (T3) and 2-fold by dioxin. Co-exposure to T3 and TCDD boosted both responses, further inducing cyp1A6 by 75% and klf9 about 60%. Additional canonical targets of each receptor, including trβa and trβb (TR) and udpgt1a (AHR) responded similarly. Induction of TH targets by TCDD in XLK-WG cells predicts that exposure could speed metamorphosis. We tested this hypothesis in two remodeling events: tail resorption and hind limb growth. Resorption of ex vivo cultured tails was accelerated by 10 nM T3, while a modest increase in resorption by 100 nM TCDD lacked statistical significance. Hind limbs doubled in length over four days following 1 nM T3 treatment, but limb length was unaffected by 100 nM TCDD. TCDD co-exposure reduced the T3 effect by nearly 40%, despite TCDD induction of klf9 in whole tadpoles, alone or with T3. These results suggest that tissue-specific TCDD effects limit or reverse the increased metamorphosis rate predicted by klf9 induction.

Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Thyroid hormone (TH) signaling regulates cell proliferation, differentiation, and metabolism. Recent studies have implicated TH signaling in cone photoreceptor viability. Using mouse models of retinal degeneration, we demonstrated that antithyroid drug treatment and targeting iodothyronine deiodinases (DIOs) to suppress cellular tri-iodothyronine (T3) production or increase T3 degradation preserves cones. In this work, we investigated the effectiveness of inhibition of the TH receptor (TR). Two genes, THRA and THRB, encode TRs; THRB2 has been associated with cone viability. Using TR antagonists and Thrb2 deletion, we examined the effects of TR inhibition. Systemic and ocular treatment with the TR antagonists NH-3 and 1-850 increased cone density by 30-40% in the Rpe65-/- mouse model of Leber congenital amaurosis and reduced the number of TUNEL+ cells. Cone survival was significantly improved in Rpe65-/- and Cpfl1 (a model of achromatopsia with Pde6c defect) mice with Thrb2 deletion. Ventral cone density in Cpfl1/Thrb2-/- and Rpe65-/- /Thrb2-/- mice was increased by 1- to 4-fold, compared with age-matched controls. Moreover, the expression levels of TR were significantly higher in the cone-degeneration retinas, suggesting locally elevated TR signaling. This work shows that the effects of antithyroid treatment or targeting DIOs were likely mediated by TRs and that suppressing TR protects cones. Our findings support the view that inhibition of TR locally in the retina is a therapeutic strategy for retinal degeneration management.-Ma, H., Yang, F., Butler, M. R., Belcher, J., Redmond, T. M., Placzek, A. T., Scanlan, T. S., Ding, X.-Q. Inhibition of thyroid hormone receptor locally in the retina is a therapeutic strategy for retinal degeneration.

Maternal Fenvalerate Exposure Induces Fetal Intrauterine Growth Restriction Through Disrupting Placental Thyroid Hormone Receptor Signaling.

Fenvalerate is an environmental endocrine disruptor that disrupts testosterone and estradiol synthesis. Nevertheless, whether fenvalerate disturbs placental TR signaling remains unclear. The aim of this study was to investigate whether maternal fenvalerate exposure causes fetal intrauterine growth restriction (IUGR) and to explore the role of placental thyroid hormone receptor (TR) signaling. Pregnant mice except controls were orally administered to fenvalerate (0.2, 2.0, or 20 mg/kg) daily throughout pregnancy. As expected, fetal weight was lowered in dams that were administered with 20.0 mg/kg of fenvalerate. Moreover, the rate of IUGR was elevated not only in male fetuses but also in female fetuses of dams exposed to 20.0 mg/kg of fenvalerate. Histopathology showed that the internal space of blood vessels in the labyrinth layer was smaller in placentas of mice exposed to fenvalerate. Mechanistic study found no significant difference on TT4 level in maternal serum, although TT3 level in maternal serum was slightly reduced in dams exposed to 2.0 mg/kg of fenvalerate. Interestingly, placental TRα1 and TRβ1 mRNAs were reduced in mice exposed to fenvalerate. Moreover, nuclear translocation of placental TRβ1 was suppressed in fenvalerate-exposed mice. Further analysis showed that placental Vegfα and Igf2, several target genes of TR signaling, were down-regulated in fenvalerate-exposed mice. In addition, mRNA level of placental CD36, Snat1, and Snat2, 3 nutrient transporters, were reduced in fenvalerate-exposed mice. These results suggest that maternal fenvalerate exposure induces fetal IUGR through disrupting placental TR signaling. These results provide a novel mechanistic explanation for fenvalerate-induced fetal IUGR.

Hypothyroidism modifies morphometry and thyroid-hormone receptor expression in periurethral muscles of female rabbits.

To evaluate the morphometry and thyroid-hormone receptor (TR) expression in pelvic (pubococcygeus, Pcm) and perineal (bulbospongiosus, Bsm) muscles of control and hypothyroid female rabbits. Hypothyroidism was induced administering 0.02% methimazole in the drinking water for one month. Hematoxylin-eosin stained muscle sections were used to evaluate the fiber cross-sectional area (CSA) and the number of peripheral myonuclei per fiber. Immunohistochemistry was used to calculate the proportion of TR immunoreactive nuclei per fiber. Significant differences were considered at a P ≤ 0.05. As compared to control rabbits, hypothyroidism increased the averaged fiber CSA and the myonuclei per fiber in the Bsm. Although the myonuclei number per fiber was also increased in the Pcm, the effect concerning the fiber CSA was only observed in a fraction of the Pcm fibers. Both TRα and TRβ were similarly expressed in the Pcm and Bsm. Hypothyroidism increased the expression of the TRα in the Bsm. Meanwhile, the expression of TR isoforms in the Pcm was not altered. Our findings support that the TR signaling is directly involved in morphometrical changes induced by hypothyroidism in the Pcm and Bsm. The effect of hypothyroidism on the Pcm and Bsm could be related to the different type of fiber and metabolism that these muscles have. Neurourol. Urodynam. 35:895-901, 2016. © 2015 Wiley Periodicals, Inc.

Pituitary specific retinoid-X receptor ligand interactions with thyroid hormone receptor signaling revealed by high throughput reporter and endogenous gene responses

Disruption of thyroid hormone (TH) signaling can compromise vital processes both during development and in the adult. We previously reported on high-throughput screening experiments for man-made TH disruptors using a stably integrated line of rat pituitary cells, GH3.TRE-Luc, in which a thyroid hormone receptor (TR) response element drives luciferase (Luc) expression. In these experiments, several retinoid/rexinoid compounds activated the reporter. Here we show that all-trans and 13-cis retinoic acid appear to function through the heterodimer partners of TRs, retinoid-X receptors (RXRs), as RXR antagonists abrogated retinoid-induced activation. The retinoids also induced known endogenous TR target genes, showing good correlation with Luc activity. Synthetic RXR-specific agonists significantly activated all tested TR target genes, but interestingly, retinoid/rexinoid activation was more consistent between genes than the extent of T3-induced activation. In contrast, the retinoids neither activated the Luc reporter construct in transient transfection assays in the human hepatocarcinoma cell line HuH7, nor two of the same T3-induced genes examined in pituitary cells. These data demonstrate the suitability and sensitivity of GH3.TRE-Luc cells for screening chemical compound libraries for TH disruption and suggest that the extent of disruption can vary on a cell type and gene-specific bases, including an underappreciated contribution by RXRs.

Abstract 5282: The role of T3-regulated microRNAs in human hepatoma cells.

Abstract Thyroid hormone (T3)/thyroid hormone receptor (TR) signaling regulates cellular growth, development and differentiation, which play important roles in vivo. T3/TR signaling promotes invasion while suppressing proliferation of hepatoma cells. MicroRNAs (miRNA) are small, short, endogenously expressed non-coding RNAs that negatively regulate gene expression. Aberrant miRNA expression has been observed in various tumor types, including hepatocellular carcinoma. At present, it is unclear whether T3/TR signaling regulates miRNAs in hepatoma cells. In our laboratory, miRNA profiling analysis using the high-throughput stem-loop RT-qPCR method revealed upregulation of miR-21 by T3/TR. A native thyroid hormone response element was identified in the primary miR-21 promoter region using promoter and chromatin immunoprecipitation assays. Overexpression of miR-21 promoted hepatoma cell migration and invasion, similar to that observed with T3 stimulation in hepatoma cells. In addition, anti-miR-21-induced suppression of cell migration was rescued by T3. T-cell lymphoma invasion and metastasis 1 (Tiam1), involved in cell migration and invasion, was identified as a miR-21 target, and downregulated by T3. Tiam1 was upregulated upon knockdown of miR-21, and conversely, downregulated upon miR-21 overexpression. Further, knockdown of Tiam1 enhanced migration and invasion may through increase of vimentin and MMP2 expression in hepatoma cells. The correlation among T3/TR, miR-21 and Tiam1 was observed in vivo is similar with in vitro. These findings indicate that miR-21 is directly upregulated by T3. Moreover, T3-mediated enhancement of hepatoma cell migration and invasion may through miR-21-targeted downregulation of Tiam1. Citation Format: Ya-Hui Huang, Yang-Hsiang Lin, Ching-Ying Chen, Kwang-Huei Lin. The role of T3-regulated microRNAs in human hepatoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5282. doi:10.1158/1538-7445.AM2013-5282

Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions

Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone's central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1.

A population of hypothalamic thermosensitive neurons regulating cardiovascular functions

Thyroid hormone is known for its profound direct effects on cardiovascular function and metabolism; however, recent studies demonstrated that the hormone also regulates these systems indirectly through the autonomic nervous system. While some of the mechanisms underlying the hormone's central effects on metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here we describe a novel population of neurons in the hypothalamus that require thyroid hormone receptor signaling for their development. Stereotaxic ablation of these cells in the mouse using a conditionally neurotoxic virus results in hypertension and temperature dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibit intrinsic temperature sensitivity in patch clamping experiments, thus providing a new connection between cardiovascular function and core temperature. Taken together, we have discovered a previously uncharacterized hypothalamic cell population potentially important for understanding hypertension, and identified developmental hypothyroidism as a novel epigenetic risk factor for cardiovascular disorders.

Identification of CCR4 and other essential thyroid hormone receptor co-activators by modified yeast synthetic genetic array analysis

Identification of thyroid hormone receptor (TR) co-regulators has enhanced our understanding of thyroid hormone (TH) action. However, it is likely that many other co-regulators remained unidentified, and unbiased methods are required to discover these proteins. We have previously demonstrated that the yeast Saccharomyces cerevisiae is an excellent system in which to study TR action, and that defined TR signaling complexes in a eukaryotic background devoid of complicating influences of mammalian cell co-regulators can be constructed and analyzed for endogenous yeast genes, many of which are conserved in mammals. Here, a modified synthetic genetic array analysis was performed by crossing a yeast strain that expressed TRbeta1 and the co-activator GRIP1/SRC2 with 384 yeast strains bearing deletions of known genes. Eight genes essential for TH action were isolated, of which 4 are conserved in mammals. Examination of one, the yeast CCR4 and its human homolog CCR4/NOT6 (hCCR4), confirmed that (i) transfected CCR4 potentiates a TH response in cultured cells more efficiently than established TR co-activators and (ii) knockdown of CCR4 expression strongly inhibited a TH response (>80%). TH treatment promoted rapid and sustained hCCR4 recruitment to the TH-responsive deiodinase 1 promoter and TR co-localizes with hCCR4 in the nucleus and interacts with hCCR4 in 2-hybrid and pull-down assays. These findings indicate that a modified yeast synthetic genetic array strategy is a feasible method for unbiased identification of conserved genes essential for TR and other nuclear receptor hormone functions in mammals.

Human testicular orphan receptor 4 enhances thyroid hormone receptor signaling

The thyroid hormone receptor (TR) and human testicular orphan receptor 4 (TR4) belong to the nuclear hormone receptor superfamily. They are ligand-dependent transcription factors. TR and TR4 bind to a similar thyroid response element (TRE), known as a direct repeat with four nucleotide spacing (DR4). This study examined the possible interaction or cross-talking between those two receptors. We hypothesized that protein-protein interaction between TR4 and TR may promote TR-mediated transcriptional activity. Glutathione S-transferase pull-down and immunoprecipitation assays showed direct interaction between TR and TR4. Electrophoretic mobility-shift assay demonstrated that TR and TR4 could co-occupy the same TRE. The interaction between TR4 and TR may enhance regulation of genes targeted by TR, such as furin, fibrinogen, cdk2 and p21 expression. We found that TR4 function is similar with TR as TR4 alone could regulate expression of some TR target genes, and could increase cell migration or inhibit cell proliferation. Importantly, the TR-dependent inhibition of cell proliferation and stimulation of cell migration are more enhanced in the HepG2-TR cells stably over-expressing TR4. Overall, TR4 not only has modulation abilities similar to TR but also can cross-talk with TR and promote the TR signaling pathway.

Anti-thyroid hormone activity of bisphenol A, tetrabromobisphenol A and tetrachlorobisphenol A in an improved reporter gene assay

Previously, we transiently transfected Gal4-fused thyroid hormone receptor (TR) expressing vector and the Gal4 response reporter structure pUAS-tk-luc into HepG2 cell, developed a TR beta-1 mediated reporter gene assay to screen for compounds that acted on the TR signaling pathway. In this study, we improved the test efficiency by changing the transfected cell line into CV-1 cell. Triiodothyronine (T3) and thyroxine (T4) induced higher luciferase expression, with the median effective concentration (EC 50) of 1.16 × 10 −8 and 1.36 × 10 −7 M, respectively. Bisphenol A (BPA) was selected as a positive antagonist, exhibiting weak anti-thyroid hormone activity with the median inhibitory concentration (IC 50) of 1.64 × 10 −5 M. The assay showed acceptable repeatability to T3 with inter coefficient of variability (CV) of 27.5% and intra CV of 18.6%. Two flame retardants, tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), were tested for their agonist and antagonist activities. As a result, we found that both of them possessed TR antagonist activity and neither of them showed agonist activity. These results suggested that TBBPA and TCBPA could act as TH antagonists. This assay provided a useful tool for the assessment of environmental chemicals as thyroid system disruptors.