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Resistance to thyroid hormone beta (RTHβ) caused by germline mutations in genes encoding thyroid hormone receptor beta (TRβ) is a rare disorder. Little information is available regarding the clinical experience of this syndrome in Japan. We retrospectively reviewed the records of 34 patients with RTHβ (21 adult females and 13 adult males) with positive TRβ mutations identified at our division between 2000 and 2020. Of the 24 patients with available clinical history, 10 (41.7%) received inappropriate treatments such as antithyroid drugs, thyroidectomy, or radioactive iodine. Diagnostic delay and inappropriate management of RTHβ are still present in Japan. Every patient except one demonstrated thyroid hormone profiles indicative of syndrome of inappropriate secretion of thyrotropin (SITSH), characterized by a hormonal profile of hyperthyroxinemia with a non-suppressed TSH concentration. Since the most common forms of hyperthyroidism including Graves' disease feature elevated thyroid hormone levels with suppressed TSH concentrations, early diagnosis of SITSH is critical for preventing inappropriate management. One patient positive for anti-thyroglobulin antibody (Tg-Ab) and anti-thyroperoxidase antibody (TPO-Ab) showed remarkably elevated TSH (>200 μIU/mL) despite thyroid hormone concentrations within the reference ranges. At least one thyroid autoantibody (Tg-Ab, TPO-Ab, or thyrotropin receptor antibodies) was identified in 37.9% (11/29) of the patients tested. One patient developed overt Graves' disease nine years after ‍RTHβ diagnosis. These findings suggest that RTHβ is frequently comorbid with additional autoimmune thyroid disorders. ‍Further research is required to identify the most appropriate treatments for RTHβ patients who develop a second thyroid ‍disorder.

Abnormal thyroid hormone (TH) function has been observed in all components of metabolic syndrome (MetS), but the mechanisms remain unclear. Altered genomic methylation status is closely related to MetS. Our aim was to determine whether methylation regulation in TH function–related genes is involved in MetS. In a small strictly selected cohort, low TH function was observed in MetS group, as well as lower THRB promoter methylation levels in peripheral blood leukocytes in a genome-wide methylation screening by Illumina 450K beadchip. The results of beadchip assay were then confirmed by Sequenom MassARRAY. Low THRB promoter methylation levels and low TH function in MetS were confirmed in another big-size validation cohort. Lower methylation levels were associated with higher THRB expression in peripheral blood leukocytes, and altered THRB promoter methylation status influenced its promoter activity and expression. In the MetS rat models constructed by high fat and high fructose diet, lower TH function was also observed, as well as lower Thrb promoter methylation levels. Furthermore, systematic inflammation observed in MetS was found to induce decreased THRB promoter methylation levels as well as corresponding THRB expression. Additionally, oral treatment with a physiological T3 dose mitigated hypertension and insulin resistance and partially alleviated hepatic steatosis and adipocyte hypertrophy in MetS rats. Low methylation levels of THRB promoter in peripheral blood leukocytes induced by systematic inflammation were involved in low TH function in MetS, whereas low TH function deteriorates MetS. This might serve as a novel therapeutic target of MetS.

The thyroid hormone receptor beta (TRβ) is a tumor suppressor in multiple types of solid tumors, most prominently in breast and thyroid cancer. An increased understanding of the molecular mechanisms by which TRβ abrogates tumorigenesis will aid in understanding the core tumor-suppressive functions of TRβ. Here, we restored TRβ expression in the MDA-MB-468 basal-like breast cancer cell line and perform RNA-sequencing to determine the TRβ-mediated changes in gene expression and associated signaling pathways. The TRβ expressing MDA-MB-468 cells exhibit a more epithelial character as determined by principle component analysis-based iterative PAM50 subtyping score and through reduced expression of mesenchymal cytokeratins. The epithelial to mesenchymal transition pathway is also significantly reduced. The MDA-MB-468 data set was further compared with RNA sequencing results from TRβ expressing thyroid cancer cell line SW1736 to determine which genes are TRβ correspondingly regulated across both cell types. Several pathways including lipid metabolism and chromatin remodeling processes were observed to be altered in the shared gene set. These data provide novel insights into the molecular mechanisms by which TRβ suppresses breast tumorigenesis.

Curcumin has been reported to have a therapeutic effect on Alzheimer’s disease (AD), but the specific mechanism remains to be elucidated. In the present research, we aimed to investigate the effect and molecular mechanism of curcumin on AD. Mouse primary hippocampal neuron cells were treated with various concentrations of beta-amyloid 42 (Aβ42) and the results found that Aβ42 inhibited cell viability in a dose-dependent manner. Compared with 50 ng/mL Aβ42, 500 ng/mL Aβ42 could further promote cell apoptosis, reduce the ratio of Nicotinamide adenine dinucleotide (NAD(+))/Nicotinamide adenine diphosphate hydride (NADH) and Adenosine 5′-triphosphate (ATP) level, and inhibit Sirtuins 3 (SIRT3) deacetylation activity and protein expression of Thyroid hormone receptor beta (Thrb) and SIRT3. Hence, 500 ng/mL Aβ42 was used to establish a cell model of AD. Curcumin significantly reversed the inhibitory effects of Aβ42 on cell viability, SIRT3 deacetylation activity, the ratio of NAD+/NADH, ATP level and the protein expression of Thrb and SIRT3, and the promotive effect on apoptosis. ChIPBase was used to predict the binding region of Thrb and SIRT3. Dual luciferase reporter gene and Chromatin immune precipitation (ChIP) assays were employed to verify the relationship between Thrb and promoter of SIRT3 mRNA. Overexpression of Thrb recovered Aβ42 induced metabolic dysfunction, while Thrb silence aggravated Aβ42 induced metabolic dysfunction. Moreover, Thrb silence or 3-TYP (a selective inhibitor of SIRT3) treatment abolished the amelioration of curcumin on Aβ42 induced metabolic dysfunction. Additionally, curcumin attenuated memory deficits in Amyloid precursor protein transgenic (APPTG) mice. Collectively, curcumin alleviated Aβ42-induced neuronal metabolic dysfunction through increasing Thrb expression and SIRT3 activity and improved cognition in APPTG mice.

Growing numbers of synthetic chemicals have potential endocrine-disrupting effects and cause potential ecological and health risks. However, the primary toxicity pathways and mechanisms of endocrine disruption are poorly understood and the existing risk assessment relies heavily on animal testing. Database mining, omics technology, and computer simulation can serve as an alternative approach to explore the mechanisms by building adverse outcome pathways (AOPs). The present study took a case of thyroid interference with triphenyl phosphate (TPP) to explain the potential toxic effects at levels from submolecules to cells utilizing the AOP framework developed by multiple techniques. This study retrieved the data from a comparative toxicogenomics database (CTD) and screened out the core gene. Molecular dynamics (MD) analysis was used to explore configuration changes and confirm the molecular initiating event (MIE). The transcriptomic analysis was further utilized to supplement the relationships between MIEs and key events (KEs) of the AOP. The thyroid hormone receptor beta (THRB) was identified as the core gene at submolecular levels. MD analysis found that the configuration changes of C-terminal helix 12 (H12) of thyroid hormone receptor beta (TR beta) were discovered as the MIE. The transcriptomic analysis extended the related KE1 at the subcellular level, such as changes in gene expression levels for coding cycle regulation (CCND1), inflammatory response (IL1A and IL6), and cell proliferation and apoptosis (BAD, TP53, and CASP9). Then, the KE2 at cellular levels such as apoptosis, cell cycle control, and cell proliferation were influenced accordingly. As a result, these alterations led to thyroid disorder as adverse outcomes. This study provided an efficient way to facilitate the complement of possible AOPs and brought new insights into understanding the toxic mechanisms of emerging synthetic chemicals.

Thyroid hormone receptors (TRs) play a critical role in human development, growth, and metabolism. Antagonists of TRs offer an attractive strategy to treat hyperthyroidism without the disadvantage of a delayed onset of drug action. While it is challenging to examine the atomistic behavior of TRs in a laboratory setting, computational methods such as molecular dynamics (MD) simulations have proven their value to elucidate ligand-induced conformational changes in nuclear receptors. Here, we performed MD simulations of TRα and TRβ complexed to their native ligand triiodothyronine (T3) as well as several antagonists. Based on the examination of 27 μs MD trajectories, we showed how binding of these compounds influences various structural features of the receptors including the helicity of helices 3 and 10 as well as the location of helix-12. Helices 3 and 12 are known to mediate coactivator association required for downstream signaling, suggesting these changes to be the molecular basis for TR antagonism. A mechanistic analysis of the trajectories revealed an allosteric pathway between H3 and H12 to be responsible for the conformational adaptations. Even though a mechanistic understanding of conformational adaptations triggered by TR antagonists is important for the development of novel therapeutics, they have not been previously examined in detail as it was done here.

Background Post-translational modification (PTM) of proteins controls various cellular functions of transcriptional regulators and participates in diverse signal transduction pathways in cancer. The thyroid hormone (triiodothyronine, T3) plays a critical role in metabolic homeostasis via its direct interaction with the thyroid hormone receptor beta (TRβ). TRβ is involved in physiological processes, such as cell growth, differentiation, apoptosis, and maintenance of metabolic homeostasis through transcriptional regulation of target genes. Objective This study was performed to characterize the specific PTM of TRβ is an active control mechanism for the proteasomal degradation of TRβ in transcriptional signaling pathways in hepatocellular carcinoma cells. Methods Based on a previous study, we predicted that the lysine methyltransferase and methylation sites of TRβ by comparing the amino acid sequences of histone H3 and TRβ. Methyl-acceptor site of TRβ was confirmed by point mutation. TRβ protein stability was evaluated by ubiquitination assay with MG132. For glucose starvation, HepG2 cells were incubated in media without D-glucose. Proliferation-related proteins were detected by western blotting. MicroRNA level and autophagy marker were measured by real-time qPCR. Results The presence of enhancer of zeste homolog 2 (Ezh2), a methyltransferase of H3 lysine 27, as a methyltransferase of TRβ also revealed that direct lysine methylation and consequent stimulated protein degradation of TRβ underlies the negative correlation between Ezh2 and TRβ. Notably, glucose starvation significantly increased lysine methylation, and methylated TRβ showed further protein instability leading to an increase in the proliferation and growth of hepatocellular carcinoma cells. Conclusions TRβ functions as a tumor suppressor in various cancers; therefore, we evaluated the effect of TRβ degradation on oncogenesis during glucose starvation. These data clearly define a functional model and provide a link between metabolism and cancer by regulating methyl-dependent protein levels of tumor suppressors. Taken together, maintaining TRβ against methyl-dependent degradation is considered a possible therapeutic target for cancer progression.

The environmental and dietary pesticide exposures can cause thyroid hormones (THs) disorders, which are associated with the high incidence of thyroid diseases worldwide. The structures of diphenyl ether pesticides and their metabolites are very similar to the structure of THs. Based on this, in silico molecular simulation approaches were used to predict, screen, evaluate and identify the binding interactions of 98 diphenyl ether structure pesticides and their metabolites (DEPMs) with 10 THs related proteins in the study. The research results indicated that these DEPMs such as fluoroglycofen (FOG), rafoxanide, diclofop, ethoxyfen and difenopenten were considered to have the greater potentials to interfere with the related proteins of THs biosynthesis, blood transport, receptor binding and metabolism. And FOG can interact with thyroid hormone receptor beta (TRβ) to form non-bond interactions. Furthermore, the results of molecular dynamics simulations showed that there were strong and stable interactions between FOG and TRβ. These results suggested that the herbicide FOG was likely to disturb THs nuclear receptor. And benzene rings and hydrophobic groups might be the characteristic chemical functional groups for DEPMs to disrupt TRβ. The relevant results of this study can be used to provide references for environmental toxicology evaluation, food safety risk assessment, and formulation and revision of pesticides and their metabolites residue limits in agricultural products and food.

Clinical interpretation of thyroid labs is usually straightforward. However, there are rare instances when the atypical profile of thyroid labs warrants systematic investigation to determine the underlying cause. We report the case of a 90-year-old Caucasian male with a chronic history of atrial fibrillation with chronic pacemaker dependence who presented with significantly elevated free thyroxine level (>7.77 ng/dL) but normal thyroid-stimulating hormone level (2.15 µIU/mL). After ruling out pituitary tumors and artifactual errors due to lab interference, the diagnosis of thyroid hormone resistance was made.

BackgroundAnaplastic thyroid cancer (ATC) is one of the most lethal endocrine cancers, with an average survival time of six months after diagnosis. These aggressive tumors have very limited treatment options highlighting a need for a deeper understanding of its mechanisms for development of more effective therapies. We have previously shown that the liganded thyroid hormone receptor beta (TR{beta}) can function as a tumor suppressor and induce re-differentiation in ATC cells. We therefore tested the hypothesis that selective activation of TR{beta} with sobetirome (GC-1) could reduce the tumorigenic phenotypes of ATC cell lines and improve the efficacy of clinically relevant therapeutics. MethodsWe used a panel of four ATC cell lines with variable genetic backgrounds to assess the ability of GC-1 to reduce the aggressive phenotype. The effects of GC-1 alone or in combination with buparlisib, alpelisib, sorafenib, and palbociclib on cell growth, viability, and migration were determined and compared with the gene expression levels of selected markers. The impact of these treatments on the cancer stem cell population was assessed by tumorsphere assay. Thyroid differentiation markers were measured by gene analysis, and sodium iodide symporter (NIS) protein level and function were determined. ResultsOur results show that GC-1 alone can decrease cell viability, growth, and slow cell migration in all four ATC cell lines. In addition, GC-1 is able to further block each of these phenotypes when combined with buparlisib, alpelisib, sorafenib, or palbociclib. GC-1 alone blocks thyrosphere outgrowth in all cell lines and increases the efficacy of each of the therapeutic agents tested. GC-1 increased NIS transcript and protein levels to allow for increased iodide uptake in ATC cells. ConclusionActivation of TR{beta} with selective agonist sobetirome (GC-1) reduces the aggressive phenotype and induced re-differentiation in ATC cells and increases the efficacy of therapeutic agents that are currently used in the treatment of ATC. These results indicate that selective activation of TR{beta} not only induces a tumor suppression program de novo but enhances the effectiveness of anti-cancer agents.

Thyroid cancer is the most common endocrine malignancy, and the global incidence has increased rapidly over the past few decades. Anaplastic thyroid cancer (ATC) is highly aggressive, dedifferentiated, and patients have a median survival of fewer than 6 months. Oncogenic alterations in ATC include aberrant phosphoinositide 3 kinase (PI3K) signaling through receptor tyrosine kinase (RTK) amplification, loss of phosphoinositide phosphatase expression and function, and protein kinase B (Akt) amplification. Furthermore, the loss of expression of the tumor suppressor thyroid hormone receptor beta (TRβ) is strongly associated with ATC. TRβ is known to suppress PI3K in follicular thyroid cancer and breast cancer by binding to the PI3K regulatory subunit p85α. However, the role of TRβ in suppressing PI3K signaling in ATC is not completely delineated. Here we report that TRβ indeed suppresses PI3K signaling in ATC cell lines through unreported genomic mechanisms, including a decrease in RTK expression and an increase in phosphoinositide and Akt phosphatase expression. Furthermore, the reintroduction and activation of TRβ in ATC cell lines enables an increase in the efficacy of the competitive PI3K inhibitors LY294002 and buparlisib on cell viability, migration, and suppression of PI3K signaling. These findings not only uncover additional tumor suppressor mechanisms of TRβ but shed light on the implication of TRβ status and activation on inhibitor efficacy in ATC tumors.

Chlorophenols (CPs) have mainly been used as a biocide, wood treatment agent and a byproduct of bleaching in paper mills. They have been a topic of concern due to their wide spread and potential effects on human and wildlife. However, data on the thresholds and effects of the number of chlorine atoms on the endocrine-disrupting potential of CPs remain scarce. In this study, we adopted two in vitro models (reporter gene assays and H295R cell line) to investigate the endocrine-disrupting effects of four CPs (pentachlorophenol (PCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,4-dichlorophenol (2,4-DCP) and 2-chlorophenol (2-CP)). The molecular docking platform was adopted to further confirm the results of the in vitro assessment. Our results revealed that PCP exhibited oestrogen receptor alpha (ERα) agonistic activity at the concentration of 10−5 M and the value of REC20 was 1.9 × 10−6 M. PCP and 2, 4, 6-TCP showed anti-oestrogenic activities with a RIC20 value of 2.8 × 10−7and 2.9 × 10−6 M, respectively. Notably, only PCP exhibited thyroid hormone receptor beta (TRβ) antagonistic activity occurred at the concentration of 10−5 M, with a RIC20 value of 1.3 × 10−6 M. The oestrogenic and thyroid hormone effects of CPs may be dependent on the number of chlorine atoms. A higher number of chlorine atoms indicated the higher effect of four CPs. The results of molecular docking were consistent with the reporter gene assay. For H295R cell line assay, PCP induced the StAR upregulation, while CYP17 was downregulated in a concentration-dependent manner by PCP and 2, 4, 6-TCP.

The point mutations like I431V on thyroid hormone receptor-beta (THR-β) gene cause resistance to thyroid hormones (RTH) with the clinical diagnosis of elevated free triiodothyronine (T3) and free thyroxin (T4) but not suppressed thyroid stimulating hormone (TSH) on the blood serum. Some ultrasonographic (USG) reports of the patients with RTH show thyroid gland disorder with goiter or nodule(s) or cyst(s) and some USG reports even with RTH are normal. I431V-mutant causes more steric hindrance while binding T3 into THR-β than the native wild type THRT3. The residue on the 431-codon is dynamic in nature showing its flexibility over the course of entry and release of T3-hormone into/from the ligand binding pocket. The more increased solvent accessible surface area of I431V-mutant than that of native I431-residue makes the partial unfolding of the globular THR-β protein. The smaller height of radial distribution function between I431-mutant and T3 shows the decrease in probability of finding the atomic particles nearby T3-hormone in THRT3-MT than in THRT3-WT. The electrostatic interaction energy between native I431 and T3 is negative, but it is positive between I431V and T3. Moreover, the internal energy of I431V-mutant has been found smaller than that of native I431-residue in THRT3 systems.BIBECHANA 16 (2019) 79-91

While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb) interacts with Hsp90 to control hormonal perception and signaling. Treatment with high Triiodothyronine (T3) concentrations induced higher Hsp90 expression in mouse fibroblasts. We can show by microarray-based displacement assay, micro-scale thermophoresis, streptavidin-conjugated quantum dot (SAv-QDs) masking assay or 8-anilino-1-naphthalene sulfone (ANS) fluorescence that T3 enhanced adenosine triphosphate (ATP) binding of Hsp90, while microarray-based interaction analyses revealed that Hsp90 bound directly TRb and was released by T3. The dose-dependent binding of Hsp90 to TRb revealed an affinity of 5 nM. The affinity near the physiological range suggests that the release of TRb induced by T3 additionally influences chaperone activity of Hsp90 when T3 increased over normal concentrations. The observation is that T3 interaction with TRb and Hsp90 is an amplifier of the cellular stress response.

TSH is a member of the pituitary glycoprotein hormone (GPH) family and consists of a non-covalently linked GPH α-subunit and hormone-specific β-subunit. The GPH α- and TSH β-subunits contain a cysteine-knot motif and several N -linked glycosylation sites, both of which are important for biological activity. TSH binds to the TSHR located on the membrane of thyroid follicular cells and stimulates thyroid growth and function, and the synthesis and release of thyroid hormones (T 3 and T 4 ). The synthesis and release of TSH are stimulated by TRH, while thyroid hormones inhibit the synthesis and release of TSH through binding to thyroid hormone receptors. TSH levels are measured as part of a thyroid function test in patients suspected of hyperthyroidism and hypothyroidism. Graves’ disease is an autoimmune thyroid disorder in which the body produces antibodies to TSHR that mimic the effects of TSH.

Thyroid cancer is the most common endocrine malignancy, and the global incidence has increased rapidly over the past few decades. Anaplastic thyroid cancer (ATC) is highly aggressive, dedifferentiated, and patients have a median survival of fewer than six months. Oncogenic alterations in ATC include aberrant PI3K signaling through receptor tyrosine kinase (RTK) amplification, loss of phosphoinositide phosphatase expression and function, and Akt amplification. Furthermore, the loss of expression of the tumor suppressor thyroid hormone receptor beta (TRβ) is strongly associated with ATC. TRβ is known to suppress PI3K in follicular thyroid cancer and breast cancer by binding to the PI3K regulatory subunit p85α. However, the role of TRβ in suppressing PI3K signaling in ATC is not completely delineated. Here we report that TRβ indeed suppresses PI3K signaling in ATC through unreported genomic mechanisms including a decrease in RTK expression and increase in phosphoinositide and Akt phosphatase expression. Furthermore, the reintroduction and activation of TRβ in ATC enables an increase in the efficacy of the competitive PI3K inhibitors LY294002 and buparlisib on cell viability, migration, and suppression of PI3K signaling. These findings not only uncover additional tumor suppressor mechanisms of TRβ but shed light into the implication of TRβ status and activation on inhibitor efficacy in ATC tumors.Abstract FigureGraphical abstract

Background : The pituitary is a key target for thyroid hormone but underlying transcriptional mechanisms are poorly understood. Thyroid hormone modifies expression of hormones, including growth hormone (GH) and thyroid-stimulating hormone (TSH, thyrotropin). Wider transcriptome responses are undefined. Thyroid hormone receptor beta (TRb) encoded by THRB are expressed in the anterior pituitary and THRB mutations cause human resistance to thyroid hormone. Method: To investigate genomic genomic regulation by TRb, we derived Thrb-HAB knockin mice that express TRb protein with a tag that is biotinylated in vivo in presence of an R26-BirA allele. Specific, sensitive streptavidin pull-down facilitated Chromatin-Affinity-Purification-sequencing (ChAPseq) to identify genomic TRβ binding sites in pituitary of male mice. Hypo- and hyperthyroidism were produced using methimazole (MMI) in drinking water for 4 weeks with/without added thyroid hormone (T3) for the 4th week. Pituitaries from wild type and Thrb-KO mice were also isolated for RNA-sequencing (RNA-seq). Selected expression changes were confirmed by quantitative PCR. Epigenetic changes were determined by ChIPseq for histone acetylation and methylation and open chromatin analysis (ATAC-seq). Results: Transcriptome analysis revealed genes with statistically different expression induced by T3, including known response genes such as Tshb, Hr and Gh. Responses were impaired in Thrb-KO mice. T3 induced recruitment of TRb binding, chromatin opening and specific histone acetylation marks. Conclusion: Most T3 response genes in pituitary depend to some extent upon TRb. T3-dependent chromatin modifications indicate properties of TRb-dependent enhancer regions and a critical role for TRb in transcriptional regulation of pituitary function.

Background Endometrial cancer (EC) is the most common gynecologic cancer in women, and the incidence of EC has increased by about 1% per year in the U. S over the last 10 years. Although 5-year survival rates for early-stage EC are around 80%, certain subtypes of EC that lose nuclear hormone receptor (NHR) expression are associated with poor survival rates. For example, estrogen receptor (ER)-negative EC typically harbors a worse prognosis compared to ER-positive EC. The molecular basis for the loss of NHR expression in endometrial tumors and its contribution to poor survival is largely unknown. Furthermore, there are no tools to systematically identify tumors that lose NHR mRNA expression relative to normal tissue. The development of such an approach could identify sets of NHR-based biomarkers for classifying patients into subgroups with poor survival outcomes. Methods Here, a new computational method, termed receptLoss, was developed for identifying NHR expression loss in endometrial cancer relative to adjacent normal tissue. When applied to gene expression data from The Cancer Genome Atlas (TCGA), receptLoss identified 6 NHRs that were highly expressed in normal tissue and exhibited expression loss in a subset of endometrial tumors. Results Three of the six identified NHRs – estrogen, progesterone, and androgen receptors – that are known to lose expression in ECs were correctly identified by receptLoss. Additionally, a novel association was found between thyroid hormone receptor beta (THRB) expression loss, increased expression of miRNA-146a, and increased rates of 5-year survival in the EC TCGA patient cohort. THRB expression loss occurs independently of estrogen and progesterone expression loss, suggesting the discovery of a distinct, clinically-relevant molecular subgroup. Conclusion ReceptLoss is a novel, open-source software tool to systematically identify NHR expression loss in cancer. The application of receptLoss to endometrial cancer gene expression data identified THRB, a previously undescribed biomarker of survival in endometrial cancer. Applying receptLoss to expression data from additional cancer types could lead to the development of biomarkers of disease progression for patients with any other tumor type. ReceptLoss can be applied to expression data from additional cancer types with the goal of identifying biomarkers of differential survival.

The flatfish, Solea senegalensis has considerable scientific interest and commercial value. The metamorphosis in this species occurs between 12 and 19 days after hatching and it takes about 1 week to complete. Eleven Bacterial Artificial Chromosomes (BAC) clones containing the various candidate genes involved in the process of metamorphosis: thyroxine 5 deiodinase 3 (dio3); forkhead box protein E4 (foxe4); melatonin receptor type 1C (mel1c); calsequestrin 1b (casq1b); thyrotropin subunit beta (tshβ); thyrotropin-releasing hormone receptor 1, 2, and 3 (trhr1, trhr2, trhr3); thyroid hormone receptor α a and b (thrαa, thrαb); and thyroid hormone receptor beta (thrβ) were analyzed by multiple Fluorescence in situ Hybridization (mFISH) and Next Generation Sequencing (NGS) techniques. The mFISH technique localized the 11 BAC clones on 12 different chromosome pairs because three of them, specifically the trhr1a, trhr2 and thrβ BAC clones, showed double signals. This signal duplication indicates a duplication of the genomic region inserted within the BAC clone, which provides evidence for the Teleost-Specific Whole Genome Duplication (TS-WGD). Micro-synteny and phylogenetic analysis showed that Cynoglossus semilaevis is the nearest species to S. senegalensis and that Danio rerio is the most distant one. The tshβ BAC clone was highly conserved as the genes belonging to this BAC were located on a single chromosome in all the species studied. These genes participate in proliferation, migration and cell-death, which are key processes during metamorphosis. Overall, micro-synteny analysis showed that most candidate genes are found in conserved genomic surroundings.

Thyroid hormones (THs) elicit significant effects on numerous physiological processes, such as growth, development, and metabolism. A lack of thyroid hormones is not compatible with normal health. Most THs effects are mediated by two different thyroid hormone receptor (TR) isoforms, namely TRα and TRβ, with the TRβ isoform known to be responsible for the main beneficial effects of TH on liver. In brain, despite the crucial role of TRα isoform in neuronal development, TRβ has been proposed to play a role in the remyelination processes. Consequently, over the past two decades, much effort has been applied in developing thyroid hormone analogs capable of uncoupling beneficial actions on liver (triglyceride and cholesterol lowering) and central nervous system (CNS) (oligodendrocyte proliferation) from deleterious effects on the heart, muscle and bone. Sobetirome (GC-1) and subsequently Eprotirome (KB2115) were the first examples of TRβ selective thyromimetics, with Sobetirome differing from the structure of thyronines because of the absence of halogens, biaryl ether oxygen, and amino-acidic side chain. Even though both thyromimetics showed encouraging actions against hypercholesterolemia, non-alcoholic steatohepatitis (NASH) and in the stimulation of hepatocytes proliferation, they were stopped after Phase 1 and Phase 2–3 clinical trials, respectively. In recent years, advances in molecular and structural biology have facilitated the design of new selective thyroid hormone mimetics that exhibit TR isoform-selective binding, and/or liver- and tissue-selective uptake, with Resmetirom (MGL-3196) and Hep-Direct prodrug VK2809 (MB07811) probably representing two of the most promising lipid lowering agents, currently under phase 2–3 clinical trials. More recently the application of a comprehensive panel of ADME-Toxicity assays enabled the selection of novel thyromimetic IS25 and its prodrug TG68, as very powerful lipid lowering agents both in vitro and in vivo. In addition to dyslipidemia and other liver pathologies, THs analogs could also be of value for the treatment of neurodegenerative diseases, such as multiple sclerosis (MS). Sob-AM2, a CNS- selective prodrug of Sobetirome has been shown to promote significant myelin repair in the brain and spinal cord of mouse demyelinating models and it is rapidly moving into clinical trials in humans. Taken together all these findings support the great potential of selective thyromimetics in targeting a large variety of human pathologies characterized by altered metabolism and/or cellular differentiation.

Highlights • Mutations in the thyroid hormone receptor beta (THRB) lead to relative hyperthyroidism in the brain. • Electrophysiological biomarkers of performance monitoring (ERN and Pe components) show a pattern similar to ADHD in carriers of THRB mutations. • The phenotype of THRB mutation carriers is indistinguishable from ADHD with regard to performance monitoring., Resistance to thyroid hormone beta (RTHβ) is a syndrome of reduced responsiveness of peripheral tissue to thyroid hormone, caused by mutations in the thyroid hormone receptor beta (THRB). Its cognitive phenotype has been reported to be similar to attention deficit hyperactivity disorder (ADHD). This study used electrophysiological biomarkers of performance monitoring in RTHβ to contribute further evidence on its phenotypical similarity to ADHD. Twenty-one participants with RTHβ aged 18–67 years and 21 matched healthy controls performed a modified flanker task during EEG recording. The RTHβ and control groups were compared on behavioural measures and components of event related potentials (ERPs), i.e. the error related negativity (ERN), the error positivity (Pe) and P3 component. There were no significant group differences with regard to behaviour. RTHβ subjects displayed significantly reduced ERN and Pe amplitudes compared to the controls in the response-locked ERPs. In addition, we observed reduced P3 amplitudes in both congruent and incongruent trials, as well as prolonged P3 latencies in RTHβ subjects in the stimulus-locked ERPs. Our findings reveal alterations in error detection and performance monitoring of RTHβ patients, likely indicating reduced error awareness. The electrophysiological phenotype of RTHß subjects with regard to action monitoring is indistinguishable from ADHD.

Commercial selection for meat-type (broiler) chickens has produced economically valuable birds with fast growth rates, enhanced muscle mass, and highly efficient feed utilization. The physiological changes that account for this improvement and unintended consequences associated with them remain largely unexplored, despite their potential to guide further advancements in broiler production efficiency. To identify effects of genetic selection on hormonal signaling in the adrenocorticotropic and thyrotropic axes, gene expression in muscle and liver and post-hatch circulating hormone concentrations were measured in legacy [Athens Canadian Random Bred (ACRB)] and modern (Ross 308) male broilers between embryonic days (e) 10 and e18 and post-hatch days (d) 10 and d40. No interactive effects or main effects of line were observed for adrenocorticotropic gene expression during either developmental period, although age effects appeared for corticosteroid-binding globulin in liver during embryogenesis and post-hatch and glucocorticoid receptor in both tissues post-hatch. There was a main line effect for circulating corticosterone, with levels in ACRB greater than those in Ross. Several thyrotropic genes exhibited line-by-age interactions during embryonic or post-hatch development. In liver, embryonic expression of thyroid hormone receptor beta was greater in ACRB on e12, and deiodinase 3 (DIO3) levels were greater in Ross on e14 and e16. In juvenile liver, deiodinase 2 (DIO2) expression was greater in ACRB on d10 but greater in Ross on d20, while DIO3 was higher in ACRB on d30 and d40. Levels of thyroid hormone receptor alpha mRNA exhibited a main line effect, with levels greater in ACRB juvenile breast muscle. Several thyrotropic genes exhibited main age effects, including DIO2 and DIO3 in embryonic breast muscle, thyroid hormone receptor alpha and thyroid hormone receptor beta in post-hatch liver, and DIO2 in post-hatch breast muscle. Circulating triiodothyronine displayed a main line effect, with levels in Ross significantly reduced as compared to ACRB. These findings suggest that in modern broilers, a decrease in levels of hormones that control basal metabolism triiodothyronine and the stress response circulating corticosterone, as well as altered expression of genes regulating thyroid hormone activity, could contribute to lower heat production, reduced stress response, and altered nutrient partitioning, leading to more efficient feed utilization and faster, more productive growth.

Bisphenol S (4-hydroxyphenyl sulfone, BPS) is increasingly used as a bisphenol A (BPA) alternative. The global usage of BPS and its analogues (BPSs) resulted in the frequent detection of their residues in multiple environmental media. We investigated their potential endocrine-disrupting effects toward thyroid hormone receptor (TR) β. The molecular interaction of BPSs toward TRβ ligand binding domain (LBD) was probed by fluorescence spectroscopy and molecular dynamics (MD) simulations. BPSs caused the static fluorescence quenching of TRβ LBD. The 100 ns MD simulations revealed that the binding of BPSs caused significant changes in the distance between residue His435 at helix 11(H11) and residue Phe459 at H12 in comparison to no ligand-bound TRβ LBD, indicating relative repositioning of H12. The recombinant two-hybrid yeast assay showed that tetrabromobisphenol S (TBBPS) and tetrabromobisphenol A (TBBPA) have potent antagonistic activity toward TRβ, with an IC10 of 10.1 and 21.1 nM, respectively. BPS and BP...

We previously reported that TRH stimulated pituitary TSHβ gene expression via an immediate increase in NR4A1 in thyrotrophs. We demonstrated that NR4A1 mRNA levels are regulated by thyroid hormone. Pituitary NR4A1 mRNA levels were decreased in mice injected with L-T4. NR4A1 promoter activity was increased by the overexpression of TRβs, and these increases were decreased by T3, and the −27∼+152 bp region was responsible for these changes in vitro . An EMSA showed the lack of TRβs-isoforms binding, and a ChIP assay demonstrated the recruitment of TRβs and NCoR in the −147∼+148 bp region in the absence of T3, whereas T3 induced their release. Experiments on the overexpression and knockdown of NCoR, and using the mutant TRs supported the involvement of NCoR in the TR-induced stimulation. These results demonstrate that thyroid hormone down-regulated basal NR4A1 mRNA levels in the pituitary, and the direct binding of TR was not required.

Purpose 3-iodothyronamine (3-T1AM), a decarboxylated and deiodinated thyroid hormone metabolite, leads at pharmacological doses to hypoinsulinemia, hyperglucagonemia and hyperglycemia in vivo. As the pancreatic Langerhans islets express thyroid hormone transmembrane transporters (THTT), we tested the hypothesis that not only plasma membrane-mediated 3-T1AM binding to and activation of G-protein coupled receptors, but also 3-T1AM metabolite(s) generated by 3-T1AM uptake and metabolism might decrease glucose-stimulated insulin secretion (GSIS). Methods Murine pancreatic β-cells MIN6 were characterized for gene expression of THTT, deiodinases and monoamine oxidases. 3-T1AM uptake and intracellular metabolism to the corresponding 3-iodothyroacetic acid were analysed by liquid-chromatography tandem mass spectrometry (LC-MS/MS) at different time points in cells as well as the conditioned medium. Mitochondrial activity, especially ATP-production, was monitored real-time after 3-T1AM application using Seahorse Bioanalyzer technique. Effect of 3-T1AM on GSIS into the culture medium was assayed by ELISA. Results MIN6 cells express classical THTT, proposed to transport 3-T1AM, as well as 3-T1AM metabolizing enzymes comparable to murine primary pancreatic islets. 3-T1AM accumulates in MIN6 cells and is metabolized by intracellular MaoB to 3-iodothyroacetic, which in turn is rapidly exported. 3-T1AM decreases mitochondrial ATP-production concentration dependently. GSIS is diminished by 3-T1AM treatment. Using LC-MS/MS, no further 3-T1AM metabolites except 3-iodothyroacetic were detectable. Conclusions This data provides a first link between cellular 3-T1AM uptake and regulation of mitochondrial energy metabolism in s-cells, resulting in reduced insulin secretion. We conclude that MIN6 is an appropriate cell model to study 3-T1AM-dependent (intra-)cellular biochemical mechanisms affecting insulin production in vitro.

Thyroid hormone, 3,3′,5-triiodo-l-thyronine (T3), mediates several physiological processes, including embryonic development, cellular differentiation and cell proliferation, via binding to its nuclear thyroid receptors (TR). Previous microarray and Chromatin immunoprecipitation (ChIP)-on-ChIP analyses have revealed that interferon-stimulated gene 20 kDa (ISG20), an exoribonuclease involved in the antiviral function of interferon, is up-regulated by T3 in HepG2-TR cells. However, the underlying mechanisms of ISG20 action in tumor progression remain unknown to date. Here, we verified induction of ISG20 mRNA and protein expression by T3 in HepG2-TR cells. Based on the ChIP-on-ChIP database, potential thyroid hormone responsive element of the ISG20 promoter region was predicted, and the result confirmed with the ChIP assay. Functional assays showed that forced expression of ISG20 leads to significant promotion of metastasis and angiogenesis, both in vitro and in vivo. Furthermore, the angiogenic-related protein, interleukin-8 (IL-8), was up-regulated through a T3-mediated increase in ISG20, as determined using a human angiogenesis array kit. Induction of IL-8 signaling activated the p-JAK2/p-STAT3 pathway, in turn, leading to promotion of tumor metastasis and angiogenesis. Furthermore, ISG20 overexpression in hepatocellular carcinoma (HCC) specimens was positively correlated with clinical parameters, including vascular invasion, α-fetoprotein and tumor size. Higher ISG20 expression was significantly correlated with poorer recurrence-free survival in HCC patients. Our results collectively indicate higher TR-dependent expression of ISG20 in a subset of HCC, supporting an oncogenic role in HCC progression.

Thyroid hormones are bound to three major serum transport proteins, thyroxin-binding globulin (TBG), transthyretin (TTR) and human serum albumin (HSA). TBG has the strongest affinity for thyroid hormones, TTR is also found in the cerebrospinal fluid and HSA is the most abundant protein in plasma. Combination defects of either a high affinity TTR or HSA variant do not compensate TBG deficiency, underscoring the dominant role of TBG among the thyroid hormone transport proteins. On the other hand, coexistence of raised affinity TTR and HSA variants causes an augmented hyperthyroxinemia. Variations in thyroid hormone transport proteins may alter thyroid function tests to mimic hypo- or hyperthyroidism. As affected individuals are clinically euthyroid and do not require treatment, identification of thyroid hormone transport protein defects is important to avoid unnecessary diagnostic and therapeutic interventions. Mammals share the multilayered system of thyroid hormone binding proteins with humans. Some of them, especially carnivores, do not express TBG. In dogs, this defect has been shown to be caused by a defective hepatocyte nuclear factor-1 binding site in the TBG promoter, preventing TBG synthesis in the liver. The major endogenous thyroid hormone metabolite 3-iodothyronamine (3-T1AM) exerts marked cryogenic, metabolic, cardiac and central nervous system actions. It is bound to apolipoproteinB-100 (ApoB100), possibly facilitating its cellular uptake via interaction with the low density lipoprotein-receptor. This review summarizes the handling of hydrophobic charged thyroid hormone signaling molecules and their metabolite 3-T1AM in aqueous body fluids and the advantages and limits of their serum distributor proteins.

The gastrointestinal tract is a well-characterized target of thyroid hormones and thyroid hormone nuclear receptors TRs, as extensively described in the literature. The paradigm is its important remodelling in amphibians during thyroid hormone-dependent metamorphosis. Interestingly, several studies have described the conservation of this hormonal signal during intestinal development in mammals. Additional data suggested that it may also play a role in intestinal homeostasis, stem cell physiology and progenitor commitment as well as in tumour development. It is worth underlining that in the mammalian intestine the functionality of the TRα1 receptor is coordinated and integrated with other signalling pathways, such as Wnt and Notch, specifically at the level of stem/progenitor cell populations. Here, we summarize these data and concepts and discuss this new role for thyroid hormones and the TRα1 receptor in the biology of intestinal epithelial precursor cells.

Thyroid hormones (THs) play key roles in modulating the overall metabolism of the body, protein synthesis, fat metabolism, neuronal and bone growth, and cardiovascular as well as renal functions. In this review, we discuss on the thyroid hormone synthesis and activation, thyroid hormone receptors (TRs) and mechanism of action, applications of thyroid hormone analogues, particularly the compounds that are selective ligands for TRβ receptors, or enzyme inhibitors for the treatment of thyroidal disorders with a specific focus on thyroid peroxidase and iodothyronine deiodinases. We also discuss on the development of small-molecule deiodinase mimetics and their mechanism of deiodination, as these compounds have the potential to regulate the thyroid hormone levels.

Objectives: Loss of estrogen and progesterone receptor expression has historically been associated with poor survival outcomes in women with uterine cancer, but the role of the remaining 46 nuclear hormone receptors remains unclear. Recently, data from The Cancer Genome Atlas demonstrated a novel association between loss of thyroid hormone receptor beta expression and increased survival in uterine cancer patients.1 The purpose of this study was to explore the association between clinical indices of thyroid function and survival outcomes in women diagnosed with uterine cancer. 1. Pique, D.G., Greally, J.M. & Mar, J.C. Identification of a novel subgroup of endometrial cancer patients with loss of thyroid hormone receptor beta expression and improved survival. BMC Cancer 20:857 (2020). Methods: A single-institution retrospective cohort study was performed examining all women diagnosed with uterine cancer from 2006-2016. Data regarding patient demographics, medical co-morbidities, histology, stage and treatment course were abstracted from the medical record. Historical records and thyroid function studies (TSH, T3, free T4) were used to identify patients who were euthyroid, hypothyroid, or hyperthyroid within 1 year prior to their cancer diagnosis until the end of the follow-up period. The primary outcome was time to death from cancer diagnosis. A Cox proportional hazards model was used to identify the association between thyroid dysfunction and survival by using thyroid status as a time-dependent covariate and adjusting for age, grade and stage of disease. Download : Download high-res image (177KB) Download : Download full-size image Results: A total of 1201 patients were included for analysis. One thousand thirty (86%) patients had no thyroid dysfunction at time of cancer diagnosis, 145 patients (12%) carried the baseline diagnosis of hypothyroidism and 26 patients (2%) had hyperthyroidism. Twenty three women with normal thyroid function at baseline developed thyroid dysfunction during follow-up (91% hypothyroid, 9% hyperthyroid). A total of 218 deaths were observed. There were no differences in survival among patients with hypothyroidism or hyperthyroidism compared to euthyroid patients [HR 0.72 (95% CI 0.47-1.12, p=0.15); HR 0.51 (95% CI 0.16-1.58, p=0.24) respectively]. Women diagnosed with thyroid dysfunction prior to cancer diagnosis were observed to have a 30% lower hazard of death compared to euthyroid women, although this did not achieve statistical significance [HR 0.70 (95% CI 0.46-1.07, p=0.10)]. Patients who developed thyroid dysfunction after cancer diagnosis were observed to have a 3.7 fold higher hazard of death compared to patients who remained euthyroid during followup [HR 3.70 (95% CI 1.60-8.30), p=0.01], but this did not maintain significance in models adjusting for age, grade and stage of disease. Conclusions: Thyroid dysfunction was not associated with a difference in survival in women with uterine cancer in our cohort. Timing of thyroid dysfunction relative to uterine cancer diagnosis may affect clinical outcomes. Additional studies are needed to explore the relationship of thyroid dysfunction and uterine cancer.

Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroid hormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division–related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development.

Thyroid hormones have important effects on cellular development, growth, and metabolism and are necessary for the healthy function of almost all tissues. Hyperthyroid patients with excess thyroid hormone levels experience tachycardia, fatigue, muscle wasting, and osteoporosis. However, although high thyroid hormone levels have adverse effects, efforts have been made to harness the beneficial effects, such as reduced serum low-density lipoprotein (LDL) cholesterol levels, elevated basal metabolic rate, and weight loss. Thyroid hormones interact with nuclear thyroid hormone receptors (TRs), and cholesterol levels are reduced through TRβ, whereas extrahepatic adverse actions are primarily connected to TRα. Thus, to develop a useful compound for clinical use, efforts have been focusing on developing compounds with isomer-specific functions based on the structure of thyroid hormones, i.e., thyromimetics that are liver and/or TRβ specific. In this short review, we discuss the development of the early thyromimetics that enabled, through modern molecular techniques, the progress towards improved design of TRβ-selective thyromimetics. We also address the early promise shown in human clinical trials and the current status of these drugs and other emerging compounds.

Thyroid hormone receptors (TRs) play crucial roles in vertebrates. Wild-type (WT) TRs function primarily as hormone-regulated transcription factors. A human endocrine disease, Resistance to Thyroid Hormone (RTH)-Syndrome, is caused by inheritance of mutant TRs impaired in the proper regulation of target gene expression. To better understand the molecular basis of RTH we compared the target genes regulated by an RTH-TRβ1 mutant (R429Q) to those regulated by WT-TRβ1. With only a few potential exceptions, the vast majority of genes we were able to identify as regulated by the WT-TRβ1, positively or negatively, were also regulated by the RTH-TRβ1 mutant. We conclude that the actions of R429Q-TRβ1 in RTH-Syndrome most likely reflect the reduced hormone affinity observed for this mutant rather than an alteration in target gene repertoire. Our results highlight the importance of target gene specificity in defining the disease phenotype and improve our understanding of how clinical treatments impact RTH-Syndrome.

Purpose: New therapeutic approaches are needed for patients with thyroid cancer refractory to radioiodine treatment. An inhibitor of bromodomain and extraterminal domain (BET) proteins, JQ1, shows potent antitumor effects in hematological cancers and solid tumors. To evaluate whether JQ1 is effective against thyroid cancer, we examined antitumor efficacy of JQ1 using the ThrbPV/PVKrasG12D mouse, a model of anaplastic thyroid cancer. Experimental Design: We treated ThrbPV/PVKrasG12D mice with vehicle or JQ1 at a dose of 50 mg/kg body weight/day starting at the age of 8 weeks for a 10-week period and monitored thyroid tumor progression. Results: JQ1 markedly inhibited thyroid tumor growth and prolonged survival of these mice. Global differential gene expression analysis showed that JQ1 suppressed the cMyc (hereafter referred to as Myc) transcription program by inhibiting mRNA expression of Myc, ccnd1, and other related genes. JQ1-suppressed Myc expression was accompanied by chromatin remodeling as evidenced by increased expression of histones and hexamethylene bis-acetamide inducible 1, a suppressor of RNA polymerase II transcription elongation. Analyses showed that JQ1 decreased MYC abundance in thyroid tumors and attenuated the cyclin D1–CDK4–Rb–E2F3 signaling to decrease tumor growth. Further analysis indicated that JQ1 inhibited the recruitment of BDR4 to the promoter complex of the Myc and Ccnd1 genes in rat thyroid follicular PCCL3 cells, resulting in decreased MYC expression at the mRNA and protein levels to inhibit tumor cell proliferation. Conclusions: These preclinical findings suggest that BET inhibitors may be an effective agent to reduce thyroid tumor burden for the treatment of refractory thyroid cancer. Clin Cancer Res; 23(2); 430–40. ©2016 AACR.

Thyroid hormone (TH) signaling is critical for tissue-organ development, growth, differentiation and metabolism, and in the liver, the most widely expressed TH receptor is TRβ. In a recent study of patients with nonalcoholic steatohepatitis (NASH), progressive liver fibrosis was associated with disrupted TH signaling. In a separate study, low serum triiodothyronine (T3) was associated with advanced NASH-fibrosis. These findings suggest that TH signaling may be a novel regulator of adult liver fibrogenesis. Herein, we hypothesized that the TH-TR axis modulates HSC phenotype and perturbations in TH-TR axis occur during liver injury. Methods: In vivo: Two murine liver injury models were used (6 weeks of methionine choline deficient (MCD) diet and 6 weeks carbon tetrachloride (CCL4) injection). Human tissues were obtained from explanted NASH and healthy donor livers. Liver fibrosis was assessed by Sirius Red (SR) staining, αSMA immunohistochemistry, and the hepatic hydroxyproline assay. Total liver expression of TRα and TRβ, αSMA and Collagen 1α1 mRNA were determined by qRT-PCR. In vitro: To determine whether TH modulates HSC activation, primary HSC and a HSC line were treated with transforming growth factor (TGF)-β (5 ng/ml), in the presence or absence of T3 (10 ng/ml). TRα knockdown in HSC was achieved using lentiviral-mediated shRNA (shTRα). Results were confirmed using TRα knock-out (TRαKO) mouse embryonic fibroblasts (MEF). HSC migration was assessed using the wound healing assay, western blot and qRT-PCR (αSMA, collagen 1α1, TRα and β). Hepatocytes were used as positive control for TRβ expression. Results: Total liver TRα and TRβ mRNA were downregulated by 4-fold (p < 0.05) during liver fibrogenesis in mice and humans. In HSC, TRα is the predominant TR (4 fold higher than TRβ; p < 0.05). TGF-β stimulation of HSC repressed TRα and TRβ mRNA expression, but this effect was blunted by T3. shTRα-HSC exhibited greater activation at baseline, with increased fibrogenesis markers αSMA and collagen 1α1, and enhanced responses to TGF-β (increased p-Smad2/3). shTRα-HSC also had greater response in wound-healing assay (1.3-fold, p < 0.05), which was consistent with enriched contractility/migration pathways obtained from RNA sequencing analysis. Conclusions: Liver expression of TRα and β is repressed during fibrogenesis following chronic liver injury. TRα is the predominant TR in HSC and perturbations in TH-TR levels regulate HSC phenotype via the TGF-β pathway. Future studies will be needed to determine if whether TH treatment could inhibit liver fibrosis progression.

Knowledge regarding the potential impacts of crude oil on endocrine signalling in freshwater aquatic vertebrates is limited. In this study, the expression of selected genes as biomarkers for altered endocrine signalling were studied in African clawed frog, Xenopus laevis tadpoles, and juvenile Mozambique tilapia, Oreochromis mossambicus exposed to weathered bunker and un-weathered refinery crude oil water accommodated fractions (WAFs). In addition, the expression of the aforementioned genes was quantified in X. laevis tadpoles exposed to surface water collected from the proximity of an underground oil bunker. The (anti)estrogenicity and (anti)androgenicity of crude oil, crude oil WAFs and surface water were furthermore evaluated using recombinant yeast. Thyroid hormone receptor beta expression was significantly downregulated in X. laevis in response to both oil WAF types, whereas, a further thyroid linked gene, type 2 deiodinase was upregulated in O. mossambicus exposed to a high concentration of bunker oil WAF. In addition, both WAFs altered the expression of the adipogenesis-linked peroxisome proliferator-activated receptor gamma in X. laevis. The crude oil and WAFs exhibited anti-estrogenic and anti-androgenic activity in vitro. However, O. mossambicus androgen receptor 2 was the only gene, representing the reproductive system, significantly affected by WAF exposure. Estrogenicity, anti-estrogenicity and anti-androgenicity were detected in surface water samples; however, no significant changes were observed in the expression of any of the genes evaluated in X. laevis exposed to surface water. The responses varied among the two model organisms used, as well as among the two types of crude oil. Nonetheless, our data provide evidence that crude oil pollution may lead to adverse health effects in freshwater fish and amphibians due to altered endocrine signalling. This article is protected by copyright. All rights reserved

Metastatic breast cancer is refractory to conventional therapies and is an end-stage disease. RUNX2 is a transcription factor that becomes oncogenic when aberrantly expressed in multiple tumor types, including breast cancer, supporting tumor progression and metastases. Our previous work demonstrated that the thyroid hormone receptor beta (TRβ) inhibits RUNX2 expression and tumorigenic characteristics in thyroid cells. As TRβ is a tumor suppressor, we investigated the compelling question whether TRβ also regulates RUNX2 in breast cancer. The Cancer Genome Atlas indicates that TRβ expression is decreased in the most aggressive basal-like subtype of breast cancer. We established that modulated levels of TRβ results in corresponding changes in the high levels of RUNX2 expression in metastatic, basal-like breast cells. The MDA-MB-231 triple-negative breast cancer cell line exhibits low expression of TRβ and high levels of RUNX2. Increased expression of TRβ decreased RUNX2 levels. The thyroid hormone-mediated suppression of RUNX2 is TRβ specific as TRα overexpression failed to alter RUNX2 expression. Consistent with these findings, knockdown of TRβ in non-tumor MCF10A mammary epithelial-like cells results in an increase in RUNX2 and RUNX2 target genes. Mechanistically, TRβ directly interacts with the proximal promoter of RUNX2 through a thyroid hormone response element to reduce promoter activity. The TRβ suppression of the oncogene RUNX2 is a signaling pathway shared by thyroid and breast cancers. Our findings provide a novel mechanism for TRβ-mediated tumor suppression in breast cancers. This pathway may be common to many solid tumors and impact treatment for metastatic cancers.

We investigate mutations in trβ2, a splice variant of thrb, identifying changes in function, structure, and behavior in larval and adult zebrafish retinas. Two N-terminus CRISPR mutants were identified. The first is a 6BP+1 insertion deletion frameshift resulting in a truncated protein. The second is a 3BP in frame deletion with intact binding domains. ERG recordings of isolated cone signals showed that the 6BP+1 mutants did not respond to red wavelengths of light while the 3BP mutants did respond. 6BP+1 mutants lacked optomotor and optokinetic responses to red/black and green/black contrasts. Both larval and adult 6BP+1 mutants exhibit a loss of red-cone contribution to the ERG and an increase in UV-cone contribution. Transgenic reporters show loss of cone trβ2 activation in the 6BP+1 mutant but increase in the density of cones with active blue, green, and UV opsin genes. Antibody reactivity for red-cone LWS1 and LWS2 opsin was absent in the 6BP+1 mutant, as was reactivity for arrestin3a. Our results confirm a critical role for trβ2 in long-wavelength cone development., Author summary There are four cone photoreceptors responsible for color vision in zebrafish: red, green, blue, and UV. The thyroid hormone receptor trβ2 is localized in the vertebrate retina. We know that it is necessary for the development of cones expressing long-wavelength-sensitive opsins (red cones), but here we investigate the functional alterations that accompany a loss of trβ2. Our work contributes to the ongoing investigations of retinal development and the involvement of thyroid hormone receptors. As suggested by previous morphological findings, fish became red colorblind when trβ2 was knocked out, and the contributions of the other three cone types shifted. Our work highlights the plasticity of photoreceptor patterning as we see changes in opsin peaks and cone sensitivity, increases in contributions of UV cones, and an attempt at a mosaic pattern in the adult retina, all in the absence of trβ2 and red cones. We now have an increased understanding of mechanisms underlying retinal development.

Although triiodothyronine (T3) induces several beneficial effects on lipid metabolism, its use is hampered by toxic side-effects, such as tachycardia, arrhythmia, heart failure, bone and muscle catabolism and mood disturbances. Since the α isoform of thyroid hormone receptors (TRs) is the main cause of T3-related harmful effects, several efforts have been made to develop selective agonists of the β isoform that could induce some beneficial effects (i.e. lowering triglyceride and cholesterol levels reducing obesity and improving metabolic syndrome), while overcoming most of the adverse T3-dependent side effects. Herein, we describe the drug discovery process sustained by ADME-Toxicity analysis that led us to identify novel agonists with selectivity for the isoform TRβ and an acceptable off-target and absorption, distribution metabolism, excretion and toxicity (ADME-Tox) profile. Within the small series of compounds synthesized, derivatives 1 and 3, emerge from this analysis as “potentially safe” to be engaged in preclinical studies. In in vitro investigation proved that both compounds were able to reduce lipid accumulation in HepG2 and promote lipolysis with comparable effects to those elicited by T3, used as reference drug. Moreover, a preliminary in vivo study confirmed the apparent lack of toxicity, thus suggesting compounds 1 and 3 as new potential TRβ-selective thyromimetics.

Context The two major forms of circulating thyroid hormones (THs) are T3 and T4. T3 is regarded as the biologically active hormone because it binds to TH receptors (TRs) with greater affinity than T4. However, it is currently unclear what structural mechanisms underlie this difference in affinity. Objective Prompted by the identification of a novel M256T mutation in a resistance to TH (RTH)α patient, we investigated Met256 in TRα1 and the corresponding residue (Met310) in TRβ1, residues previously predicted by crystallographic studies in discrimination of T3 vs T4. Methods Clinical characterization of the RTHα patient and molecular studies (in silico protein modeling, radioligand binding, transactivation, and receptor–cofactor studies) were performed. Results Structural modeling of the TRα1-M256T mutant showed that distortion of the hydrophobic niche to accommodate the outer ring of ligand was more pronounced for T3 than T4, suggesting that this substitution has little impact on the affinity for T4. In agreement with the model, TRα1-M256T selectively reduced the affinity for T3. Also, unlike other naturally occurring TRα mutations, TRα1-M256T had a differential impact on T3- vs T4-dependent transcriptional activation. TRα1-M256A and TRβ1-M310T mutants exhibited similar discordance for T3 vs T4. Conclusions Met256-TRα1/Met310-TRβ1 strongly potentiates the affinity of TRs for T3, thereby largely determining that T3 is the bioactive hormone rather than T4. These observations provide insight into the molecular basis for underlying the different affinity of TRs for T3 vs T4, delineating a fundamental principle of TH signaling., Met256-TRα1/Met310-TRβ1 determine the differential bioactivity of T3 vs T4, providing the molecular basis for the clinical concept that T4 functions as prohormone and T3 as bioactive hormone.

Leucine 341 has been predicted from crystal structure as an important residue for thyroid hormone receptor beta (TRβ) function, but this has never been confirmed in functional studies. Here, a novel p.L341V mutation as a cause of resistance to TRβ is described, suggesting an important role for L341 in TRβ function. In silico and in vitro studies confirmed that substituting L341 with valine and other non-polar amino acids impairs sensitivity of TRβ for triiodothyronine to various degrees, depending on their side-chain size and orientation.

Resistance to thyroid hormone beta (RTHβ) is a syndrome characterized by reduced responsiveness of peripheral tissues to thyroid hormone (TH). In most cases, the disorder is associated with germline pathogenic variants in the thyroid hormone receptor beta (THRB) gene. This paper summarizes the clinical and biochemical presentation of the disease, providing a comprehensive overview on molecular genetic features. Particular care is given in reporting all identified THRB variants with an assessed or unknown clinical significance. Our aim is to offer a useful tool for clinical and genetic specialists in order to ease clinical diagnosis and genetic counseling.

Anaplastic thyroid cancer (ATC) is among the most lethal human cancers, with an average survival time of six months. These tumors are characterized by rapid local extension, resistance to radioactive iodine therapy and mainstream chemotherapy, and distant metastasis. There are very limited treatment options for this aggressive form of thyroid cancer, highlighting a need for a deeper understanding of its mechanisms for development of new targeted therapies. Loss of expression of the thyroid hormone receptor beta (TRβ) is correlated with aggressive disease and decreased survival in other human cancer types, including breast cancer, hepatocellular carcinoma, and melanoma, suggesting a tumor suppressor role1-3. Decreased TRβ expression is observed in patients with ATC via tissue microarray4 and in representative ATC cell lines5. To test the hypothesis that rescue of TRβ can restore a tumor suppressor program and a more differentiated phenotype in ATC cells, we re-introduced constitutive TRβ expression in the SW1736 ATC cell line by stable lentiviral transduction. Proliferation assays reveal that SW1736 cells expressing TRβ have a 30% decrease in proliferation rate compared to empty vector control cells. This prompted us to examine the differences in the transcriptome between our SW1736 cells expressing TRβ and the empty vector control cells to determine the potential mechanisms underlying this observation. Establishment of TRβ expression restored a transcriptional response to thyroid hormone treatment, in concordance with our proliferation studies. Upon transcript quantitation and downstream pathway analysis, we observed changes in several pathways that are well known to influence thyroid cancer growth, including mTOR, PTEN, and TGFβ signalling. Upstream regulators of this transcriptional response included many epigenetic regulators, most notably BRG1, KDM5B, and BRD4. This suggests that restoration of TRβ signalling results in a ligand-dependent chromatin remodeling response to facilitate the gene expression changes we observed. In addition, we measured changes in markers of thyroid differentiation by qPCR and found that TRβ expression and treatment with thyroid hormone increased expression of several key genes including TPO, DIO1, and NIS. In summary, our data support a role for TRβ as a tumor suppressor and mediator of thyroid differentiation in thyroid cells. Importantly, our analyses will direct further investigation into targets and pathways that can be exploited as therapeutic strategies in ATC. 1. Martinez-Iglesias, O. et al.Cancer Res69, 501-509, (2009). 2. Aranda, A. et al. Trends in endocrinology and metabolism: TEM20, 318-324, (2009). 3. Suzuki, H., et al. Thyroid12, 963-969, (2002). 4. Landa, I. et al.J Clin Invest126, 1052-1066, (2016). 5. Carr, F. E. et al.Endocrinology157, 3278-3292, (2016).

Integrin αVβ3 is a structural protein of the plasma membrane that transduces signals from extracellular matrix proteins and has recently been shown to contain a novel receptor for thyroid hormone. Thyroid hormone signals are converted by αVβ3 into mitogen-activated protein kinase (MAPK) (ERK1/2) activation and downstream intracellular events in the cell nucleus. The latter include post-translational modification of the nuclear thyroid hormone receptor (TRβ1) and complex cellular or tissue responses, such as hormone-induced angiogenesis via basic fibroblast growth factor release. The integrin receptor for thyroid hormone has been shown to mediate proliferative effects of the hormone on certain tumor cell lines, including murine glioma/glioblastoma cells and human breast cancer (MCF-7) cells. More than one mechanism may account for this hormonal action, but in vitro studies indicate a direct hormonal action on cellular proliferation. Other possible mechanisms involve indirect actions via the release of tumor growth factors and effects on cell migration. In the intact organism, support of tumor growth by thyroid hormone is postulated to include angiogenesis. Crosstalk between the integrin thyroid hormone receptor and the epidermal growth factor receptor on the plasma membrane may be another mechanism by which thyroid hormone may modify tumor cell growth. Tetraiodothyroacetic acid (tetrac) is an iodothyronine analog that has no agonist activity at the integrin receptor, but inhibits binding of l-thyroxine and 3,5,3´-triiodo-l-thyronine to the receptor, preventing MAPK activation and consequent actions downstream of MAPK. In vitro studies and a preliminary in vivo experiment indicate that tetrac blocks the action of thyroid hormone on tumor cell proliferation. Both unmodified tetrac and tetrac reformulated as a nanoparticle that does not gain access to the cell interior are under investigation in animal models as anticancer agents. Also under study is the susceptibility of other human cancer cell lines to induction of proliferation by physiological concentrations of thyroid hormone.

Thyroid hormone nuclear receptors (TRs) mediate thyroid hormone’s activities in growth, differentiation, and development. Two TR genes (α and β ) encode four thyroid hormone-binding receptors that regulate target gene expression. Mutations of the TRβ gene cause the genetic syndrome of resistance to thyroid hormone. Studies indicate a close association between TRβ mutations and several human cancers, suggesting their oncogenic role. A TRβ gene knock-in mutant mouse (TRβPV/PV mouse) that spontaneously develops thyroid cancer allows elucidation of the oncogenic functions in vivo. TRβPV is a potent dominant negative mutant identified in a resistance to thyroid hormone patient. Molecular studies indicate that the PV mutant mediates its oncogenic activities via nucleus-initiated transcription and novel extranuclear actions. Thus, the deleterious effects of the gene mutations go beyond resistance to thyroid hormone and are more severe and extensive than previously envisioned. This newly identified oncogene exert...

Thyroid hormone (TH) action is crucial for the development of several tissues. A number of syndromes are associated with reduced responsiveness to thyroid hormones, expanding the original definition of thyroid hormone resistance, firstly described by Refetoff and collaborators in 1967, which is characterized by elevated circulating levels of T4 and T3 with measurable serum TSH concentrations, as a consequence of mutations of thyroid hormone receptor beta (TRβ). Recently, other forms of insensitivity to TH have been identified: defects in cell surface transporters such as the monocarboxylate transporter 8 (MCT8), genetic disorders of thyroid hormone metabolism due to alterations of selenoprotein synthesis, which comprise the deiodinase enzymes, and finally, mutations in the thyroid hormone receptor alpha (TRα). Since the syndromes of TH insensitivity are characterized by extremely variable clinical manifestations and different molecular mechanisms, this group of thyroid disorders has been very recently reclassified and a novel nomenclature proposed.

Syndromes with impaired sensitivity to thyroid hormone (TH) include three types of resistance to thyroid hormone (RTH) syndromes (RTHβ, RTHα, and nonTR-RTH) and also include patients with defects in TH transport into cells (thyroid hormone cell membrane transport defect, THCMTD) and defects in TH metabolism (thyroid hormone metabolism defect, THMD). The overall goal of this chapter is to discuss the treatment options for patients with various syndromes of impaired sensitivity to TH. These are rare conditions with little or no evidence-based information for the “best” treatment of patients. The first step toward treatment of all of these syndromes is making an accurate diagnosis based on a combination of the clinical presentation and laboratory tests that ultimately requires genetic confirmation. Recognizing the etiology leads to a more logical approach to treatment.