Thyroid Hormone Receptor Action Research Articles

Functions and Mechanism of Thyroid Hormone Receptor Action During Amphibian Development.

Thyroid hormones and their receptors (TRs) play critical roles during vertebrate development. One of the most dramatic developmental processes regulated by thyroid hormones is frog metamorphosis, which mimics the postembryonic (perinatal) period in mammals. Here, we review some of the findings on the developmental functions of thyroid hormones and TRs as well as their associated mechanisms of action obtained from this model system. More than 2 decades ago, a dual function model was proposed for TR in anuran development. During larval development, unliganded receptors recruit corepressors to repress thyroid hormone response genes to prevent premature metamorphic changes. Subsequently, when thyroid hormone levels rise, liganded receptors recruit coactivators to activate thyroid hormone response genes, leading to metamorphic changes. Over the years, molecular and genetic approaches have provided strong support for this model and have shown that it is applicable to mammalian development as well as to understanding the diverse effects of thyroid hormones in normal physiology and diseases caused by thyroid hormone signaling dysfunction.

Canonical and Noncanonical Contribution of Thyroid Hormone Receptor Isoforms Alpha and Beta to Cardiac Hypertrophy and Heart Rate in Male Mice.

Background: Stimulation of ventricular hypertrophy and heart rate are two major cardiac effects of thyroid hormone (TH). The aim of this study was to determine invivo which TH receptor (TR)-α or β-and which mode of TR action-canonical gene expression or DNA-binding independent noncanonical action-mediate these effects. Methods: We compared global TRα and TRβ knockout mice (TRαKO; TRβKO) with wild-type (WT) mice to determine the TR isoform responsible for T3 effects. The relevance of TR DNA binding was studied in mice with a mutation in the DNA-binding domain that selectively abrogates DNA binding and canonical TR action (TRαGS; TRβGS). Hearts were studied with echocardiography at baseline and after 7 weeks of T3 treatment. Gene expression was measured with real-time polymerase chain reaction. Heart rate was recorded with radiotelemetry transmitters for 7 weeks in untreated, hypothyroid, and T3-treated mice. Results: T3 induced ventricular hypertrophy in WT and TRβKO mice, but not in TRαKO mice. Hypertrophy was also induced in TRαGS mice. Thus, hypertrophy is mostly mediated by noncanonical TRα action. Similarly, repression of Mhy7 occurred in WT and TRαGS mice. Basal heart rate was largely dependent on canonical TRα action. But responsiveness to hypothyroidism and T3 treatment as well as expression of pacemaker gene Hcn2 were still preserved in TRαKO mice, demonstrating that TRβ could compensate for absence of TRα. Conclusions: T3-induced cardiac hypertrophy could be attributed to noncanonical TRα action, whereas heart rate regulation was mediated by canonical TRα action. TRβ could substitute for canonical but not noncanonical TRα action.

Identification by Liquid Chromatography-Tandem Mass Spectrometry and Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry of the Contributor to the Thyroid Hormone Receptor Agonist Activity in Effluents from Sewage Treatment Plants.

3,3',5-Triiodothyroacetic acid (TRIAC) was identified as a major contributor to the activity of thyroid hormone receptor (TR) agonists in environmental water. TRIAC contributed 60-148% of the TR-agonist activity in effluents from sewage treatment plants (STPs). Meanwhile, the contributions of 3,5,3'-triiodothyronine (T3), 3,3',5,5'-tetraiodothyronine (T4), and analogues were <1%. TRIAC concentrations in the range of 0.30-4.2 ng/L are likely enough to cause disruption of the thyroid system in living aquatic organisms. The origin of TRIAC in the STP effluents was investigated by analyzing both STP influents and effluents. Relatively high concentrations of T3 and T4 (2.5 and 6.3 ng/L, respectively) were found only in the influents. TRIAC was identified only in the effluents. These findings suggested that T3 and T4 in STP influents were potentially converted into TRIAC during activated sludge treatment or by other means. The evaluation of TRIAC at relevant environmental concentrations by in vivo assays and an appropriate treatment to reduce the TR activity in sewage are needed.

Selective thyroid hormone receptor beta agonist, GC-1, is capable to reduce growth of colorectal tumor in syngeneic mouse models

Objective The effect of thyroid hormone (TH) on cancers was proposed more than 100 years ago; however, conclusions are conflicting. THs are precisely regulated at tissue and cellular levels. It seems that this regulation is altered in cancers. Thyroid hormone receptor beta (TRβ) has anti-proliferative and tumor-suppressive effects in many cancer cells. Therefore, we decided to investigate thyroid hormone receptor beta (THRB) expression and activation by the selective agonist, GC-1, on tumor growth in a syngeneic mouse model of colorectal cancer (CRC) and colon cell lines. Methods In vitro cell viability assay using MTT analysis, cell cycle analysis by PI staining, and FACS analysis were performed. In vivo tumor growth measurements were carried out by caliper and [18F] Fluoro-2-deoxy-2-D-glucose (FDG) – PET imaging. Gene expressions were determined using quantitative-PCR. Results Some concentrations of GC-1 had a marked negative effect on the cell viability of colorectal cell lines. Cell cycle analysis showed that the anti-proliferative effect of GC-1 may not result from cell cycle arrest or apoptosis. Tumor growth analysis in mice harboring colorectal tumor showed that GC-1 treatment for 8 d profoundly inhibited tumor growth and 18FDG uptake. THRB expression was decreased in mice tumor; however, it was upregulated following GC-1 administration. Conclusions Our results showed that specific activation of TRβ by GC-1 had negative effect on tumor growth and restored its gene expression in tumors of CRC mice model.

Hyperthyroidism exacerbates ischemic reperfusion injury in the kidney.

Thyroid hormones are critical regulators of vertebrate development and metabolism. Under hyperthyroid conditions, excess thyroid hormones induce expression of several enzymes and activities via activation of ligand-bound thyroid hormone receptors (TRs). Arginase (ARG) is downstream of a ligand-bound TR and overexpression of ARG2 induces the production of reactive oxygen species and subsequent exacerbation of kidney ischemia/reperfusion (I/R) injury. To clarify the association between I/R-induced kidney injury and hyperthyroidism, mice were pretreated with L-thyroxine (LT4) or vehicle alone, then subjected to I/R. Proximal tubular cell-specific conditional knockout of thyroid hormone receptor β (TRβcKO) mice was generated and the effects of I/R were analyzed. Hyperthyroidism enhanced tubular damage and fibrosis in the kidneys of mice after I/R. Hyperthyroidism induced tubular cell necroptosis following inflammatory cell accumulation in the kidney after I/R. ARG2 expressions and reactive oxygen species accumulated in the kidneys of hyperthyroid mice after I/R, but these changes were ameliorated in the kidneys of TRβcKO mice. Hyperthyroidism-enhanced kidney injury was ameliorated in the kidney of TRβcKO mice after I/R. These results suggest that excess thyroid hormones are disadvantageous for the kidney under ischemic stress. Overt hypothyroidism represents a severe thyroid hormone deficiency disease that requires LT4 treatment, while overreplacement or iatrogenic thyrotoxicosis might cause kidney injury.

Synergistic gene regulation by thyroid hormone and glucocorticoid in the hippocampus.

The hippocampus is considered the center for learning and memory in the brain, and its development and function is greatly affected by the thyroid and stress axes. Thyroid hormone (TH) and glucocorticoids (GC) are known to have a synergistic effect on developmental programs across several vertebrate species, and their effects on hippocampal structure and function are well-documented. However, there are few studies that focus on the processes and genes that are cooperatively regulated by the two hormone axes. Cross-regulation of the thyroid and stress axes in the hippocampus occurs on multiple levels such that TH can regulate the expression of the GC receptor (GR) while GC can modulate tissue sensitivity to TH by controlling the expression of TH receptor (TR) and enzymes involved in TH biosynthesis. Thyroid hormone and GC are also known to synergistically regulate the transcription of genes associated with neuronal function and development. Synergistic gene regulation by TH and GC may occur through the direct, cooperative action of TR and GR on common target genes, or by indirect mechanisms involving gene regulatory cascades activated by TR and GR. In this chapter, we describe the known physiological effects and underlying molecular mechanisms of TH and GC synergistic gene regulation in the hippocampus.

Main Factors Involved in Thyroid Hormone Action

The thyroid hormone receptors are the mediators of a multitude of actions by the thyroid hormones in cells. Most thyroid hormone activities require interaction with nuclear receptors to bind DNA and regulate the expression of target genes. In addition to genomic regulation, thyroid hormones function via activation of specific cytosolic pathways, bypassing interaction with nuclear DNA. In the present work, we reviewed the most recent literature on the characteristics and roles of different factors involved in thyroid hormone function in particular, we discuss the genomic activity of thyroid hormone receptors in the nucleus and the functions of different thyroid hormone receptor isoforms in the cytosol. Furthermore, we describe the integrin αvβ3-mediated thyroid hormone signaling pathway and its rapid nongenomic action in the cell. We furthermore reviewed the thyroid hormone transporters enabling the uptake of thyroid hormones in the cell, and we also include a paragraph on the proteins that mediate thyroid receptors’ shuttling from the nucleus to the cytosol.

The Role of Thyroid Hormone in the Regulation of Cerebellar Development.

The proper organized expression of specific genes in time and space is responsible for the organogenesis of the central nervous system including the cerebellum. The epigenetic regulation of gene expression is tightly regulated by an intrinsic intracellular genetic program, local stimuli such as synaptic inputs and trophic factors, and peripheral stimuli from outside of the brain including hormones. Some hormone receptors are expressed in the cerebellum. Thyroid hormones (THs), among numerous circulating hormones, are well-known major regulators of cerebellar development. In both rodents and human, hypothyroidism during the postnatal developmental period results in abnormal morphogenesis or altered function. THs bind to the thyroid hormone receptors (TRs) in the nuclei and with the help of transcriptional cofactors regulate the transcription of target genes. Gene regulation by TR induces cell proliferation, migration, and differentiation, which are necessary for brain development and plasticity. Thus, the lack of TH action mediators may directly cause aberrant cerebellar development. Various kinds of animal models have been established in a bid to study the mechanism of TH action in the cerebellum. Interestingly, the phenotypes differ greatly depending on the models. Herein we summarize the actions of TH and TR particularly in the developing cerebellum.

Cells to Systems Screening for Thyroid Hormone Disrupting Chemicals in the Brain

Precisely timed and regulated thyroid hormone (TH) levels are essential for vertebrate development and metabolism. To detect environmental chemicals that disrupt TH action, we developed an integrated luciferase reporter cell line in rat pituitary GH3 cells that is TH responsive through activation of endogenous thyroid hormone receptors (TRs). High throughput screening as part of the Tox21 program identified several non-TH related positive hits as RXR agonists, a supposed “silent” heterodimer partner for TRs. These data showed RXR agonists can affect TR signaling at least in pituitary cells. We extended these results to our sensitive and specific in vivo model for TH action, Xenopus laevis metamorphosis. We previously demonstrated that RXR agonists like organotins and the pharmaceutical bexarotene, strongly potentiate (and RXR antagonists inhibit) TH induced metamorphic programs, at the morphological, cellular (e.g. apoptosis or proliferation), transgenic reporter gene levels, and transcriptomic responses in tadpole tails. We have now extended this analysis to include RNA-Seq experiments over distinct time points in the tadpole brain, a common target of TH in humans and frogs, revealing specific gene sets particularly affected by TH and RXR ligands working in concert. Very few genes were affected by RXR ligands alone. The remarkable overlap between the environmental toxicant tributyltin and the synthetic and specific RXR ligand bexarotene regulated transcriptomes provides strong evidence that they have a common molecular target in multiple tissues. We have also created germline mutations in all RXR family members in Xenopus tropicalis (alpha, beta and gamma), and both copies of the duplicated TR beta gene in Xenopus laevis via genome editing approaches. This will allow us to further investigate TR-RXR heterodimer function across tissues and developmental timeframes, and in response to known and suspected TR and RXR ligands. Our studies revealed an unanticipated degree of TR and RXR ligand interactions in vitro and in vivo, highlighting a surprising role of RXRs as avenues for TH endocrine disruption, including the brain.

IPSC-Derived Human Hepatocytes as a Novel Model to Investigate Thyroid Hormone Action and Signaling

Thyroid hormone (TH) actions are essential to normal metabolism and neurologic function. Abnormal TH levels can lead to significant morbidity including metabolic abnormalities, cardiac disease, and obesity. TH action is mediated by the thyroid hormone receptor (TR) isoforms and their coregulators. In the disease models of Resistance to Thyroid Hormone (RTH) it is clear that the TR isoforms play tissue-specific roles in humans, however a platform for drug discovery to potentially treat the diseases does not exist. We hypothesize that human hepatocytes (iHeps) derived from induced pluripotent stem cells (iPSCs) will serve as an ideal model to study TH signaling and to delineate mechanisms of TH diseases in humans. To investigate the TH action in humans, we have developed a serum free human iPSC hepatic differentiation protocol that utilizes sequential exposure to growth factors to mimic actual human development. iHeps are robustly responsive to TH with over 1000 regulated genes. Importantly, they allow us to use CRISPR/Cas9 to determine molecular physiology and mechanism of disease. To demonstrate the role of TRs in TH signaling in human liver cells we have focused on TRβ1 the main TR isoform expressed in liver. we knocked out TRβ1 (THRBKO) using guide RNAs (gRNAs) we engineered to specific genomic loci to truncate the expression of this isoform at the DNA binding domain. Also, to define mechanisms of TR action in the context of RTH, we used this same approach to introduce the Δ337T mutation found in human TRβ1 that causes RTH. Both THRBKO and Δ337T lines are karyotypically normal and flow cytometry analysis in both lines demonstrated that these lines differentiated normally into hepatocytes after gene editing with CRISPR/Cas9. Once clones were identified the edited iHeps were treated with PBS (vehicle) or T3 (10nM) for 24 hours. Real-time quantitative PCR (RT qPCR) was done to assess mRNA expression of T3 target genes. RT qPCR analysis confirmed the success of TRβ1 deletion as the response to T3 was lost on both positive and negative THRB targets. In the Δ337T iHeps, the response to TH was diminshed indicating the successful generation of this line. These preliminary results confirm our ability to edit IPSCs and then to differentiate into hepatocytes, allowing us to further study the action of TH and the mutations involved in RTH.

Activation of thyroid hormone receptor‐β improved disease activity and metabolism independent of body weight in a mouse model of non‐alcoholic steatohepatitis and fibrosis

Activation of hepatic thyroid hormone receptor ß (THR-ß) is associated with systemic lipid lowering, increased bile acid synthesis and fat oxidation. In patients with non-alcoholic steatohepatitis (NASH), treatment with THR-ß agonists led to reduction in hepatic steatosis and circulating lipids, and resolution of NASH. We chose resmetirom (MGL-3196), a liver-directed, selective THR-ß agonist, as a prototype to investigate the effects of THR-ß agonism in mice with diet-induced obesity (DIO) and biopsy-confirmed advanced NASH with fibrosis. C57Bl/6J mice were fed a diet high in fat, fructose and cholesterol for 34 weeks, and only biopsy-confirmed DIO-NASH mice with fibrosis were included. Resmetirom was then administered at a daily dose of 3 mg/kg p.o. over a period of eight weeks. Systemic and hepatic metabolic parameters, histological NAFLD activity and fibrosis scores, and liver RNA expression profiles were determined to assess the effect of THR-ß agonism. Treatment with resmetirom did not influence body weight but led to significant reduction (p<0.05) in liver weight (-43 %), hepatic steatosis (-53 %), plasma ALT activity (-49 %), liver and plasma cholesterol (-27 % and -60 %, respectively), and blood glucose (6.3 vs. 7.5 mmol/l). These metabolic effects translated into significant improvement in NAFLD activity score. Moreover, lower α-smooth muscle actin content and down-regulation of genes involved in fibrogenesis indicated a decrease in hepatic fibrosis. Our model robustly reflected clinical observations of body weight-independent improvements in systemic and hepatic metabolism including anti-steatotic activity.

Investigation of NH3 as a selective thyroid hormone receptor modulator in larval zebrafish (Danio rerio)

Thyroid hormones (THs) are essential for normal vertebrate development and diverse environmental chemicals are hypothesized to cause developmental toxicity by disrupting TH-mediated signaling. The larval zebrafish (Danio rerio) is an emerging in vivo model of developmental TH disruption; however, the effects of TR antagonism have not yet been studied in zebrafish. NH3, generally considered a potent and specific thyroid hormone receptor (TR) antagonist, has been used in rodents and Xenopus laevis to characterize phenotypes of TR antagonism. The objective of this study is to determine the effects of NH3 on endpoints previously determined to be TH-sensitive in larval zebrafish, specifically teratology and mortality, photomotor behavior, and mRNA expression of TH signaling genes. Zebrafish embryos were exposed to NH3 via static waterborne exposure at concentrations ranging from 0.001 to 10 μM beginning at 6 h post-fertilization (hpf) through 5 days post fertilization (dpf). Significant mortality and teratogenesis was observed at 3, 4, and 5 dpf in zebrafish exposed to NH3 at 10 μM. At concentrations that did not cause significant mortality, NH3 did not exert a consistent antagonistic effect on photomotor behavior assays or mRNA expression when administered alone or in the presence of exogenous T4. Rather, depending on the NH3 concentration and larval age NH3 decreased or increased swimming triggered by transition from light to dark. Similarly, inconsistent antagonistic and agonistic effects on mRNA expression of TH signaling genes were noted following treatment with NH3 alone. NH3 did inhibit T4 (30 nM)-induced gene expression; however, this was only consistently observed at a concentration of NH3 (10 μM) that also caused significant mortality. Collectively, these results suggest that NH3 does not act solely as a TR antagonist in larval zebrafish, but instead exhibits complex modulatory effects on TR activity. These data support the hypothesis that NH3 is a selective thyroid hormone receptor modulator. Further studies of NH3 interactions with the zebrafish thyroid hormone receptor are required to characterize the activity of NH3 in target tissues of the larval zebrafish at the molecular level, highlighting the importance of characterizing NH3 effects in specific models of TH-disruption to better interpret its actions in mechanistic screens of environmental chemicals for TH action.

Regulation of gene transcription by thyroid hormone receptor β agonists in clinical development for the treatment of non-alcoholic steatohepatitis (NASH)

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRβ agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRβ-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRβ and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRβ agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

Noncanonical Action of Thyroid Hormone Receptors α and β.

Thyroid hormone (TH) is essential for the regulation of many physiological processes, especially growth, organ development, energy metabolism and cardiovascular effects. TH acts via the TH receptors (TR) α and β. By binding to thyroid hormone responsive elements (TREs) on the DNA, TRs regulate expression of TH target genes. Thus, TRs are mainly characterized as ligand dependent transcription factors and regulation of gene expression and protein synthesis is considered the canonical mode of TH/TR action. The demonstration that the ligand-bound TRs α and β also mediate activation of the phosphatidylinositol-3-kinase (PI3K) pathway established noncanonical TH/TR action as an additional mode of TH signaling. Recently, TR mutant mouse models allowed to determine the underlying mode of TH/TR action, either canonical or noncanonical TH/TR signaling, for several physiological TH effects in vivo: Regulation of the hypothalamic-pituitary-thyroid axis requires DNA-binding of TRβ, whereas hepatic triglyceride content appears to be regulated by noncanonical TRβ signaling. TRα mediated effects in bone development are dependent on DNA-binding, whereas several cardiovascular TRα effects are rapid and independent from DNA-binding. Therefore, noncanonical TH/TR action contributes to the overall effects of TH in physiology.

The In Vitro Functional Impairment of Thyroid Hormone Receptor Alpha 1 Isoform Mutants Is Mainly Dictated by Reduced Ligand Sensitivity.

Background: Thyroid hormone (TH) acts on TH receptors (TRs) and regulates gene transcription by binding of TRs to TH response elements (TREs) in target gene promoters. The transcriptional activity of TRs is modulated by interactions with TR-coregulatory proteins. Mutations in TRα cause resistance to thyroid hormone alpha (RTHα). In this study, we analyzed if, beyond reduced triiodothyronine (T3) affinity, altered interactions with cofactors or different TREs could account for the differential impaired transcriptional activity of different mutants. Methods: We evaluated four mutants derived from patients (D211G, M256T, A263S, and R384H) and three artificial mutants at equivalent positions in patients with RTHβ (T223A, L287V, and P398H). The in vitro transcriptional activity was evaluated on TRE-luciferase reporters (DR4, IR0, and ER6). The affinity for T3 and interaction with coregulatory proteins (nuclear receptor corepressor 1 [NCoR1] and steroid receptor coactivator 1 [SRC1]) were also determined. Results: We found that the affinity for T3 was significantly reduced for all mutants, except for TRα1-T223A. The reduction in the T3 sensitivity of the transcriptional activity on three TREs, the dissociation of the corepressor NCoR1, and the association of the coactivator SRC1 recruitment for each mutant correlated with the reduced affinity for T3. We did not observe mutation-specific alterations in interactions with cofactors or TREs. Conclusions: In summary, the degree of impaired transcriptional activity of mutants is mainly determined by their reduced affinity for T3.

Limited Chemical Structural Diversity Found to Modulate Thyroid Hormone Receptor in the Tox21 Chemical Library.

Background:Thyroid hormone receptors (TRs) are critical endocrine receptors that regulate a multitude of processes in adult and developing organisms, and thyroid hormone disruption is of high concern for neurodevelopmental and reproductive toxicities in particular. To date, only a small number of chemical classes have been identified as possible TR modulators, and the receptors appear highly selective with respect to the ligand structural diversity. Thus, the question of whether TRs are an important screening target for protection of human and wildlife health remains.Objective:Our goal was to evaluate the hypothesis that there is limited structural diversity among environmentally relevant chemicals capable of modulating TR activity via the collaborative interagency Tox21 project.Methods:We screened the Tox21 chemical library (8,305 unique structures) in a quantitative high-throughput, cell-based reporter gene assay for TR agonist or antagonist activity. Active compounds were further characterized using additional orthogonal assays, including mammalian one-hybrid assays, coactivator recruitment assays, and a high-throughput, fluorescent imaging, nuclear receptor translocation assay.Results:Known agonist reference chemicals were readily identified in the TR transactivation assay, but only a single novel, direct agonist was found, the pharmaceutical betamipron. Indirect activation of TR through activation of its heterodimer partner, the retinoid-X-receptor (RXR), was also readily detected by confirmation in an RXR agonist assay. Identifying antagonists with high confidence was a challenge with the presence of significant confounding cytotoxicity and other, non-TR-specific mechanisms common to the transactivation assays. Only three pharmaceuticals—mefenamic acid, diclazuril, and risarestat—were confirmed as antagonists.Discussion:The results support limited structural diversity for direct ligand effects on TR and imply that other potential target sites in the thyroid hormone axis should be a greater priority for bioactivity screening for thyroid axis disruptors. https://doi.org/10.1289/EHP5314

Paradigms of Dynamic Control of Thyroid Hormone Signaling.

Thyroid hormone (TH) molecules enter cells via membrane transporters and, depending on the cell type, can be activated (i.e., T4 to T3 conversion) or inactivated (i.e., T3 to 3,3'-diiodo-l-thyronine or T4 to reverse T3 conversion). These reactions are catalyzed by the deiodinases. The biologically active hormone, T3, eventually binds to intracellular TH receptors (TRs), TRα and TRβ, and initiate TH signaling, that is, regulation of target genes and other metabolic pathways. At least three families of transmembrane transporters, MCT, OATP, and LAT, facilitate the entry of TH into cells, which follow the gradient of free hormone between the extracellular fluid and the cytoplasm. Inactivation or marked downregulation of TH transporters can dampen TH signaling. At the same time, dynamic modifications in the expression or activity of TRs and transcriptional coregulators can affect positively or negatively the intensity of TH signaling. However, the deiodinases are the element that provides greatest amplitude in dynamic control of TH signaling. Cells that express the activating deiodinase DIO2 can rapidly enhance TH signaling due to intracellular buildup of T3. In contrast, TH signaling is dampened in cells that express the inactivating deiodinase DIO3. This explains how THs can regulate pathways in development, metabolism, and growth, despite rather stable levels in the circulation. As a consequence, TH signaling is unique for each cell (tissue or organ), depending on circulating TH levels and on the exclusive blend of transporters, deiodinases, and TRs present in each cell. In this review we explore the key mechanisms underlying customization of TH signaling during development, in health and in disease states.

Number, Proliferative Activity, and Expression of Thyroid Hormone Receptors in Dermal Fibroblasts in Mice with Changed Thyroid Status.

We studied the intensity of age-specific changes in the dermis (number and proliferative activity of fibroblasts) in mice with normal and experimentally changed level of thyroid hormones. Receptors of thyroid hormones, TR-α and TR-β, in mouse dermal fibroblasts were identified by immunohistochemical methods. The relative expression of Thra, Thrb, and Dio2 genes was assessed by real-time PCR analysis. From the second to fifth month of life, the number of fibroblasts in the connective tissue layer of mouse skin decreased by 42.3%. The number of fibroblasts in the dermis of 5-month-old mice treated with Thyrozol significantly decreases by 25.9% (p<0.05), and vice versa, in mice receiving thyroxin this parameter increased by 4.7% in comparison with the control (p>0.05). TR-α and TR-β were identified in dermal fibroblasts in all groups of mice. No differences in the content TR-α and Thra gene expression in 2- and 5-month-old mice of the control and experimental were revealed. TR-β content in dermal fibroblasts of 2-month-old animals was maximum and exceeded this value in 5-month-old control mice by 25%. The number of these receptors decreased by 33.3% in mice treated with Thyrozol and increased by 25% in animals receiving thyroxin injection in comparison with the control. Relative expression of Thrb gene significantly increased only in mice treated with thyroxin. Comparative analysis of the relative expression of Dio2 gene revealed no differences between the experimental and control groups. Changes in the level of thyroid hormones, content of TR-β, and relative Thrb gene expression contribute to agerelated shifts in the number and proliferative activity of mouse dermal fibroblasts.

Thyroid hormone receptor function in maturing ovine cardiomyocytes.

Plasma thyroid hormone (tri-iodo-l-thyronine; T3 ) concentrations rise near the end of gestation and is known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for the action of thyroid hormone in fetal cardiomyocytes. Loss of thyroid hormone receptor (TR)α1 abolishes T3 signalling via extracellular signal-related kinase and Akt in fetal cardiomyocytes. The expression of TRα1 and TRβ1 in ovine fetal myocardium increases with age, although TRα1 levels always remain higher than those of TRβ1. Near term fetal cardiac myocytes are more sensitive than younger myocytes to thyroid receptor blockade by antagonist, NH3, and to the effects of TRα1/α2 short interfering RNA. Although T3 is known to abrogate ovine cardiomyocyte proliferation stimulated by insulin-like growth factor 1, this effect is mediated via the genomic action of thyroid hormone receptors, with little evidence for non-genomic mechanisms. We have previously shown that the late-term rise in tri-iodo-l-thyronine (T3 ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T3 -induced changes are mediated via thyroid hormone receptors (TRs) or by non-genomic mechanisms. Fetal cardiomyocytes were isolated from 102±3 and 135±1days of gestational age (dGA) sheep (n=7 per age; term ∼145dGA). Cells were treated with T3 (1.5nm), insulin-like growth factor (IGF)-1 (1μgmL-1 ) or a combination in the presence of TR antagonist NH3 (100nm) or following short interfering RNA (siRNA) knockdown of TRα1/α2. Proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) uptake (10μm). Western blots measured protein levels of extracellular signal-related kinase (ERK), Akt, TRα1/β1 and p21. Age specific levels of TRα1/β1 were measured in normal hearts from fetuses [95dGA (n=8), 135dGA (n=7)], neonates (n=8) and adult ewes (n=7). TRα1 protein levels were consistently >50% more than TRβ1 at each gestational age (P<0.05). T3 reduced IGF-1 stimulated proliferation by ∼50% in 100dGA and by ∼75% in 135dGA cardiomyocytes (P<0.05). NH3 blocked the T3 +IGF-1 reduction of BrdU uptake without altering the phosphorylation of ERK or Akt at both ages. NH3 did not suppress T3 -induced p21 expression in 100dGA cardiomyocytes in 135dGA cardiomyocytes, NH3 alone reduced BrdU uptake (-28%, P<0.05), as well as T3 -induced p21 (-75%, P<0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T3 +IGF-1 reduction of BrdU uptake and dramatically reduced ERK and Akt signalling in 135dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T3 signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.

Protein Disulfide Isomerase Modulates the Activation of Thyroid Hormone Receptors.

Thyroid hormone receptors (TRs) are responsible for mediating thyroid hormone (T3 and T4) actions at a cellular level. They belong to the nuclear receptor (NR) superfamily and execute their main functions inside the cell nuclei as hormone-regulated transcription factors. These receptors also exhibit so-called “non-classic” actions, for which other cellular proteins, apart from coregulators inside nuclei, regulate their activity. Aiming to find alternative pathways of TR modulation, we searched for interacting proteins and found that PDIA1 interacts with TRβ in a yeast two-hybrid screening assay. The functional implications of PDIA1—TR interactions are still unclear; however, our co-immunoprecipitation (co-IP) and fluorescence assay results showed that PDI was able to bind both TR isoforms in vitro. Moreover, T3 appears to have no important role in these interactions in cellular assays, where PDIA1 was able to regulate transcription of TRα and TRβ-mediated genes in different ways depending on the promoter region and on the TR isoform involved. Although PDIA1 appears to act as a coregulator, it binds to a TR surface that does not interfere with coactivator binding. However, the TR:PDIA1 complex affinity and activation are different depending on the TR isoform. Such differences may reflect the structural organization of the PDIA1:TR complex, as shown by models depicting an interaction interface with exposed cysteines from both proteins, suggesting that PDIA1 might modulate TR by its thiol reductase/isomerase activity.