Canonical Thyroid Hormone Receptor ß Action Stimulates Hepatocyte Proliferation in Male Mice.

CONTEXT: 3,5,3'-L-triiodothyronine (T3) is a potent inducer of hepatocyte proliferation via the Wnt/ß-catenin signaling pathway. Previous studies suggested the involvement of rapid noncanonical thyroid hormone receptor (TR) ß signaling, directly activating hepatic Wnt/ß-catenin signaling independent from TRß DNA binding. However, the mechanism by which T3 increases Wnt/ß-catenin signaling in hepatocytes has not yet been determined. OBJECTIVE: We aimed to determine whether DNA binding of TRß is required for stimulation of hepatocyte proliferation by T3. METHODS: Wild-type (WT) mice, TRß knockout mice (TRß KO), and TRß mutant mice with either specifically abrogated DNA binding (TRß GS) or abrogated direct phosphatidylinositol 3 kinase activation (TRß 147F) were treated with T3 for 6 hours or 7 days. Hepatocyte proliferation was assessed by Kiel-67 (Ki67) staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Activation of ß-catenin signaling was measured in primary murine hepatocytes. Gene expression was analyzed by microarray, gene set enrichment analysis (GSEA), and quantitative reverse transcription polymerase chain reaction. RESULTS: T3 induced hepatocyte proliferation with an increased number of Ki67-positive cells in WT and TRß 147F mice (9.2% ±â€…6.5% and 10.1% ±â€…2.9%, respectively) compared to TRß KO and TRß GS mice (1.2% ±â€…1.1% and 1.5% ±â€…0.9%, respectively). Microarray analysis and GSEA showed that genes of the Wnt/ß-catenin pathway-among them, Fzd8 (frizzled receptor 8) and Ctnnb1 (ß-catenin)-were positively enriched only in T3-treated WT and TRß 147F mice while B-cell translocation gene anti-proliferation factor 2 was repressed. Consequently, expression of Ccnd1 (CyclinD1) was induced. CONCLUSIONS: Instead of directly activating Wnt signaling, T3 and TRß induce key genes of the Wnt/ß-catenin pathway, ultimately stimulating hepatocyte proliferation via CyclinD1. Thus, canonical transcriptional TRß action is necessary for T3-mediated stimulation of hepatocyte proliferation.

Thyroid hormone receptor and ERRα coordinately regulate mitochondrial fission, mitophagy, biogenesis, and function.

Thyroid hormone receptor ß1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and ß-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA, ULK1, DRP1, or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.

Thyroid Hormone Receptor ß Suppression of RUNX2 Is Mediated by Brahma-Related Gene 1-Dependent Chromatin Remodeling.

Thyroid hormone receptor ß (TRß) suppresses tumor growth through regulation of gene expression, yet the associated TRß-mediated changes in chromatin assembly are not known. The chromatin ATPase brahma-related gene 1 (BRG1; SMARCA4), a key component of chromatin-remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenesis and TRß-mediated gene expression is unknown. We previously identified the oncogene runt-related transcription factor 2 (RUNX2) as a repressive target of TRß. Here, we report differential expression of BRG1 in nonmalignant and malignant thyroid cells concordant with TRß. BRG1 and TRß have similar nuclear distribution patterns and significant colocalization. BRG1 interacts with TRß, and together, they are part of the regulatory complex at the RUNX2 promoter. Loss of BRG1 increases RUNX2 levels, whereas reintroduction of TRß and BRG1 synergistically decreases RUNX2 expression. RUNX2 promoter accessibility corresponded to RUNX2 expression levels. Inhibition of BRG1 activity increased accessibility of the RUNX2 promoter and corresponding expression. Our results reveal a mechanism of TRß repression of oncogenic gene expression: TRß recruitment of BRG1 induces chromatin compaction and diminishes RUNX2 expression. Therefore, BRG1-mediated chromatin remodeling may be obligatory for TRß transcriptional repression and tumor suppressor function in thyroid tumorigenesis.

SKP2 Activation by Thyroid Hormone Receptor ß2 Bypasses Rb-Dependent Proliferation in Rb-Deficient Cells.

Germline RB1 mutations strongly predispose humans to cone precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRß2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRß1. TRß2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRß2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRß1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRß2 counteracts TRß1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838-50. ©2017 AACR.

Effects of hyper- and hypothyroidism on the development and proliferation of testicular cells in prepubertal rats.

Thyroid hormones are important in the development and regulation of testes. This study was conducted to determine the effects of hyper- and hypothyroidism on testicular development in prepubertal rats aged 20-70 days. Weaning male rats (20 days old) until day 70 age were randomly divided into four groups: control, hyperthyroid (hyper-T), hypothyroid (hypo-T) and hypothyroid treated with thyroxine (T4) (hypo-T+T4). The results indicated that thyroid hormones caused a significant effect in body and testis weights, and food and water consumption. In addition there were changes in serum concentrations of tri-iodothyronine, T4, thyroid stimulating hormone (TSH) and testosterone. Histomorphology showed a significant decrease in seminiferous tubule diameter in hyper-T compared to the other groups. Leydig cell numbers showed a significant elevation in hyper-T but not in hypo-T groups. Immunostaining indicated that TSH receptor (TSHR), thyroid hormone receptors α/ß (TRαß) and proliferating cell nuclear antigen (PCNA) have the roles in testicular development. Our findings suggest that hyper- and hypo-thyroidism regulate testicular cell proliferation and spermatogenesis in prepubertal rats, indicating that expression of TSHR, TRαß and PCNA may be regulated by thyroid hormones that are involved in testicular development; and that the administration of T4 to the hypo-T+T4 group leads to an improvement in the testicular condition.

Thyroid Hormone Receptor-ß (TRß) Mediates Runt-Related Transcription Factor 2 (Runx2) Expression in Thyroid Cancer Cells: A Novel Signaling Pathway in Thyroid Cancer.

Dysregulation of the thyroid hormone receptor (TR)ß is common in human cancers. Restoration of functional TRß delays tumor progression in models of thyroid and breast cancers implicating TRß as a tumor suppressor. Conversely, aberrant expression of the runt-related transcription factor 2 (Runx2) is established in the progression and metastasis of thyroid, breast, and other cancers. Silencing of Runx2 diminishes tumor invasive characteristics. With TRß as a tumor suppressor and Runx2 as a tumor promoter, a compelling question is whether there is a functional relationship between these regulatory factors in thyroid tumorigenesis. Here, we demonstrated that these proteins are reciprocally expressed in normal and malignant thyroid cells; TRß is high in normal cells, and Runx2 is high in malignant cells. T3 induced a time- and concentration-dependent decrease in Runx2 expression. Silencing of TRß by small interfering RNA knockdown resulted in a corresponding increase in Runx2 and Runx2-regulated genes, indicating that TRß levels directly impact Runx2 expression and associated epithelial to mesenchymal transition molecules. TRß specifically bound to 3 putative thyroid hormone-response element motifs within the Runx2-P1 promoter ((-)105/(+)133) as detected by EMSA and chromatin immunoprecipitation. TRß suppressed Runx2 transcriptional activities, thus confirming TRß regulation of Runx2 at functional thyroid hormone-response elements. Significantly, these findings indicate that a ratio of the tumor-suppressor TRß and tumor-promoting Runx2 may reflect tumor aggression and serve as biomarkers in biopsy tissues. The discovery of this TRß-Runx2 signaling supports the emerging role of TRß as a tumor suppressor and reveals a novel pathway for intervention.

Thyroid hormone receptor regulates most genes independently of fibroblast growth factor 21 in liver.

Thyroid hormone (TH) acts through specific receptors (TRs), which are conditional transcription factors, to induce fibroblast growth factor 21 (FGF21), a peptide hormone that is usually induced by fasting and that influences lipid and carbohydrate metabolism via local hepatic and systemic endocrine effects. While TH and FGF21 display overlapping actions when administered, including reductions in serum lipids, according to the current models these hormones act independently in vivo. In this study, we examined mechanisms of regulation of FGF21 expression by TH and tested the possibility that FGF21 is required for induction of hepatic TH-responsive genes. We confirm that active TH (triiodothyronine (T3)) and the TRß-selective thyromimetic GC1 increase FGF21 transcript and peptide levels in mouse liver and that this effect requires TRß. T3 also induces FGF21 in cultured hepatocytes and this effect involves direct actions of TRß1, which binds a TRE within intron 2 of FGF21. Gene expression profiles of WT and Fgf21-knockout mice are very similar, indicating that FGF21 is dispensable for the majority of hepatic T3 gene responses. A small subset of genes displays diminished T3 response in the absence of FGF21. However, most of these are not obviously directly involved in T3-dependent hepatic metabolic processes. Consistent with these results, T3-dependent effects on serum cholesterol are maintained in the Fgf21(-/-) background and we observe no effect of the Fgf21-knockout background on serum triglycerides and glucose. Our findings indicate that T3 regulates the genes involved in classical hepatic metabolic responses independently of FGF21.

Vascular smooth muscle cell apoptosis is an early trigger for hypothyroid atherosclerosis.

AIMS: Endothelial dysfunction is an initial and vascular smooth muscle cell (VSMC) apoptosis, a later step of atherosclerosis. Hypothyroidism accelerates atherosclerosis. However, the early events responsible for this pro-atherosclerotic effect are unclear. METHODS AND RESULTS: Rats were resistant to induction of atherosclerosis by high cholesterol diet alone, but became susceptible in hypothyroid state achieved by administration of propylthiouracil (PTU) for 6 weeks. VSMC dysfunction and apoptosis were obvious within 1 week after PTU treatment, without signs of endothelial dysfunction. This early VSMC damage was caused by hypothyroidism but not the high cholesterol diet. In ApoE knockout mice, PTU-induced hypothyroidism triggered early VSMC apoptosis, increased oxidative stress, and accelerated atherosclerosis development. Thyroid hormone supplementation (T4, 10, or 50 µg/kg) prevented atherogenic phenotypes in hypothyroid rats and mice. In rats, thyroidectomy caused severe hypothyroidism 5 days after operation, which also led to rapid VSMC dysfunction and apoptosis. In vitro studies did not show a direct toxic effect of PTU on VSMCs. In contrast, thyroid hormone (T3, 0.75 µg/L plus T4, 50 nmol/L) exerted a direct protection against VSMC apoptosis, which was reduced by knockdown of TRα1, rather than TRß1 and TRß2 receptors. TRα1-mediated inhibition of apoptotic signalling of JNKs and caspase-3 contributed to the anti-apoptotic action of thyroid hormone. CONCLUSION: These findings provide an in vivo example for VSMC apoptosis as an early trigger of hypothyroidism-associated atherosclerosis, and reveal activation of TRα1 receptors to prevent VSMC apoptosis as a therapeutic strategy in this disease.

Inhibition of tumorigenesis by the thyroid hormone receptor ß in xenograft models.

BACKGROUND: Previous studies showed a close association between several types of human cancers and somatic mutations of thyroid hormone receptor ß (TRß) and reduced expression of TRß due to epigenetic inactivation and/or deletion of the THRB gene. These observations suggest that TRß could act as a tumor suppressor in carcinogenesis. However, the mechanisms by which TRß could function to inhibit tumorigenesis are less well understood. METHODS: We used the human follicular thyroid cancer cell lines (FTC-133 and FTC-236 cells) to elucidate how functional expression of the THRB gene could affect tumorigenesis. We stably expressed the THRB gene in FTC cells and evaluated the effects of the expressed TRß on cancer cell proliferation, migration, and tumor growth in cell-based studies and xenograft models. RESULTS: Expression of TRß in FTC-133 cells, as compared with control FTC cells without TRß, reduced cancer cell proliferation and impeded migration of tumor cells through inhibition of the AKT-mTOR-p70 S6K pathway. TRß expression in FTC-133 and FTC-236 led to less tumor growth in xenograft models. Importantly, new vessel formation was significantly suppressed in tumors induced by FTC cells expressing TRß compared with control FTC cells without TRß. The decrease in vessel formation was mediated by the downregulation of vascular endothelial growth factor in FTC cells expressing TRß. CONCLUSIONS: These findings indicate that TRß acts as a tumor suppressor through downregulation of the AKT-mTOR-p70 S6K pathway and decreased vascular endothelial growth factor expression in FTC cells. The present results raise the possibility that TRß could be considered as a potential therapeutic target for thyroid cancer.

Oncogenic Actions of the Nuclear Receptor Corepressor (NCOR1) in a Mouse Model of Thyroid Cancer.

Studies have suggested that the nuclear receptor corepressor 1 (NCOR1) could play an important role in human cancers. However, the detailed molecular mechanisms by which it functions in vivo to affect cancer progression are not clear. The present study elucidated the in vivo actions of NCOR1 in carcinogenesis using a mouse model (Thrb(PV/PV) mice) that spontaneously develops thyroid cancer. Thrb(PV/PV) mice harbor a dominantly negative thyroid hormone receptor ß (TRß) mutant (denoted as PV). We adopted the loss-of-the function approach by crossing Thrb(PV) mice with mice that globally express an NCOR1 mutant protein (NCOR1ΔID) in which the receptor interaction domains have been modified so that it cannot interact with the TRß, or PV, in mice. Remarkably, expression of NCOR1ΔID protein reduced thyroid tumor growth, markedly delayed tumor progression, and prolonged survival of Thrb(PV/PV)Ncor1 (ΔID/ΔID) mice. Tumor cell proliferation was inhibited by increased expression of cyclin-dependent kinase inhibitor 1 (p21(waf1/cip1); Cdkn1A), and apoptosis was activated by elevated expression of pro-apoptotic BCL-Associated X (Bax). Further analyses showed that p53 was recruited to the p53-binding site on the proximal promoter of the Cdkn1A and the Bax gene as a co-repressor complex with PV/NCOR1/histone deacetylas-3 (HDAC-3), leading to repression of the Cdkn1A as well as the Bax gene in thyroids of Thrb(PV/PV) mice. In thyroids of Thrb(PV/PV)Ncor1 (ΔID/ΔID) mice, the p53/PV complex could not recruit NCOR1ΔID and HDAC-3, leading to de-repression of both genes to inhibit cancer progression. The present studies provided direct evidence in vivo that NCOR1 could function as an oncogene via transcription regulation in a mouse model of thyroid cancer.

Role of thyroid hormone receptor subtypes α and ß on gene expression in the cerebral cortex and striatum of postnatal mice.

The effects of thyroid hormones (THs) on brain development and function are largely mediated by the control of gene expression. This is achieved by the binding of the genomically active T3 to transcriptionally active nuclear TH receptors (TRs). T3 and the TRs can either induce or repress transcription. In hypothyroidism, the reduction of T3 lowers the expression of a set of genes, the positively regulated genes, and increases the expression of negatively regulated genes. Two mechanisms may account for the effect of hypothyroidism on genes regulated directly by T3: first, the loss of T3 signaling and TR transactivation, and second, an intrinsic activity of the unliganded TRs directly responsible for repression of positive genes and enhancement of negative genes. To analyze the contribution of the TR subtypes α and ß, we have measured by RT-PCR the expression of a set of positive and negative genes in the cerebral cortex and the striatum of TR-knockout male and female mice. The results indicate that TRα1 exerts a predominant but not exclusive role in the regulation of positive and negative genes. However, a fraction of the genes analyzed are not or only mildly affected by the total absence of TRs. Furthermore, hypothyroidism has a mild effect on these genes in the absence of TRα1, in agreement with a role of unliganded TRα1 in the effects of hypothyroidism.

Action of specific thyroid hormone receptor α(1) and ß(1) antagonists in the central and peripheral regulation of thyroid hormone metabolism in the rat.

BACKGROUND: The iodine-containing drug amiodarone (Amio) and its noniodine containing analogue dronedarone (Dron) are potent antiarrhythmic drugs. Previous in vivo and in vitro studies have shown that the major metabolite of Amio, desethylamiodarone, acts as a thyroid hormone receptor (TR) α(1) and ß(1) antagonist, whereas the major metabolite of Dron debutyldronedarone acts as a selective TRα(1) antagonist. In the present study, Amio and Dron were used as tools to discriminate between TRα(1) or TRß(1) regulated genes in central and peripheral thyroid hormone metabolism. METHODS: Three groups of male rats received either Amio, Dron, or vehicle by daily intragastric administration for 2 weeks. We assessed the effects of treatment on triiodothyronine (T(3)) and thyroxine (T(4)) plasma and tissue concentrations, deiodinase type 1, 2, and 3 mRNA expressions and activities, and thyroid hormone transporters monocarboxylate transporter 8 (MCT8), monocarboxylate transporter 10 (MCT10), and organic anion transporter 1C1 (OATP1C1). RESULTS: Amio treatment decreased serum T(3), while serum T(4) and thyrotropin (TSH) increased compared to Dron-treated and control rats. At the central level of the hypothalamus-pituitary-thyroid axis, Amio treatment decreased hypothalamic thyrotropin releasing hormone (TRH) expression, while increasing pituitary TSHß and MCT10 mRNA expression. Amio decreased the pituitary D2 activity. By contrast, Dron treatment resulted in decreased hypothalamic TRH mRNA expression only. Upon Amio treatment, liver T(3) concentration decreased substantially compared to Dron and control rats (50%, p<0.01), but liver T(4) concentration was unaffected. In addition, liver D1, mRNA, and activity decreased, while the D3 activity and mRNA increased. Liver MCT8, MCT10, and OATP1C1 mRNA expression were similar between groups. CONCLUSION: Our results suggest an important role for TRα1 in the regulation of hypothalamic TRH mRNA expression, whereas TRß plays a dominant role in pituitary and liver thyroid hormone metabolism.

Thyroid hormone receptors control developmental maturation of the middle ear and the size of the ossicular bones.

Thyroid hormone is critical for auditory development and has well-known actions in the inner ear. However, less is known of thyroid hormone functions in the middle ear, which contains the ossicles (malleus, incus, stapes) that relay mechanical sound vibrations from the outer ear to the inner ear. During the later stages of middle ear development, prior to the onset of hearing, middle ear cavitation occurs, involving clearance of mesenchyme from the middle ear cavity while the immature cartilaginous ossicles attain appropriate size and ossify. Using in situ hybridization, we detected expression of Thra and Thrb genes encoding thyroid hormone receptors α1 and ß (TRα1 and TRß, respectively) in the immature ossicles, surrounding mesenchyme and tympanic membrane in the mouse. Thra(+/PV) mice that express a dominant-negative TRα1 protein exhibited deafness with elevated auditory thresholds and a range of middle ear abnormalities including chronic persistence of mesenchyme in the middle ear into adulthood, markedly enlarged ossicles, and delayed ossification of the ossicles. Congenitally hypothyroid Tshr(-/-) mice and TR-deficient Thra1(-/-);Thrb(-/-) mice displayed similar abnormalities. These findings demonstrate that middle ear maturation is TR dependent and suggest that the middle ear is a sensitive target for thyroid hormone in development.

Resistance to thyroid hormone is modulated in vivo by the nuclear receptor corepressor (NCOR1).

Mutations in the ligand-binding domain of the thyroid hormone receptor ß (TRß) lead to resistance to thyroid hormone (RTH). These TRß mutants function in a dominant-negative fashion to interfere with the transcription activity of wild-type thyroid hormone receptors (TRs), leading to dysregulation of the pituitary-thyroid axis and resistance in peripheral tissues. The molecular mechanism by which TRß mutants cause RTH has been postulated to be an inability of the mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone (TH)-mediated transcription activity. To test this hypothesis in vivo, we crossed Thrb(PV) mice (a model of RTH) expressing a human TRß mutant (PV) with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit a TR or a PV mutant. Remarkably, in the presence of NCOR1ΔID, the abnormally elevated thyroid-stimulating hormone and TH levels found in Thrb(PV) mice were modestly but significantly corrected. Furthermore, thyroid hyperplasia, weight loss, and other hallmarks of RTH were also partially reverted in mice expressing NCOR1ΔID. Taken together, these data suggest that the aberrant recruitment of NCOR1 by RTH TRß mutants leads to clinical RTH in humans. The present study suggests that therapies aimed at the TR-NCOR1 interaction or its downstream actions could be tested as potential targets in treating RTH.

Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock.

AIM: Non-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo. METHODS: Serum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators. RESULTS: Patients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRß (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48  h. CONCLUSIONS: Patients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

Distinct regulation of cardiac I(f) current via thyroid receptors alpha1 and beta1.

Thyroid hormone (TH) markedly modulates cardiovascular function and heart rate. The pacemaker current I(f) and encoding hyperpolarization-activated cation (HCN) genes have been identified as TH targets. To analyze the specific contribution and functional significance of thyroid receptor isoforms responsible for HCN gene transactivation, we generated transgenic neonatal rat cardiomyocytes with adenovirus-mediated overexpression of the thyroid receptors alpha1 (TR alpha 1) and beta1 (TR beta 1), and analyzed native I(f) current and expression levels of the underlying molecular components HCN2 and HCN4. Initial results revealed that spontaneous beating activity was higher in TR alpha 1- and lower in TR beta 1-expressing cardiomyocytes. This was associated with accelerated depolarization velocity and abbreviated action potential duration in cells overexpressing TR alpha 1, while TR beta 1 suppressed phase 4 depolarization and prolonged action potentials. Consistently, TR alpha 1-infected myocytes exhibited larger I(f) current densities along with increased HCN2 and HCN4 mRNA and protein levels. In contrast, HCN2 gene expression was not significantly affected by TR beta 1. TR beta 1 exclusively suppressed HCN4 transcription. T3 application led to significant effects only in controls and TR alpha 1-infected cardiomyocytes; whereas, no ligand-dependent actions were observed in TR beta 1-expressing neonatal cardiomyocytes. Our results demonstrate that TR alpha 1 and TR beta 1 divergently regulate cardiac pacing activity. TH-induced positive chronotropic effects are likely to be mediated by TR alpha 1 through enhanced expression of I(f) pacemaker current and its underlying genes.

Divergent regulation of cardiac KCND3 potassium channel expression by the thyroid hormone receptors alpha1 and beta1.

The cardiac transient outward current I(to) is regulated by thyroid hormone (T3). However, it remains unclear whether T3 directly modulates underlying gene transcription and which thyroid receptor (TR) isoform might be responsible for gene transactivation. To clarify this situation, we analysed the role of T3 and its receptors alpha1 (TRalpha1) and beta1 (TRbeta1) in regulation of KCNA4, KCND2, KCND3 and KCNIP2 transcription in rat cardiomyocytes. Initial results demonstrated a T3-mediated increase of I(to) current density. T3 stimulation enhanced KCND2 and KCND3 expression and decreased KCNA4 transcription, while KCNIP2 remained unaffected. To dissect the role of TRalpha1 and TRbeta1 in T3-dependent I(to) modulation, TRalpha1 and TRbeta1 were overexpressed in cardiomyocytes by adenovirus-mediated gene transfer. TRalpha1 increased I(to), while TRbeta1 significantly reduced I(to) in size, which was associated with TRalpha1-mediated increase and TRbeta1-mediated reduction of KCND2/3 transcription. To further evaluate a possible direct interaction of TRalpha1 and TRbeta1 with the KCND3 promoter, TR expression vectors were cotransfected with a construct containing 2335 bp of the KCND3 5'-flanking sequence linked to a luciferase reporter into ventricular myocytes. While the TRalpha1 aporeceptor enhanced KCND3 transcription, the TRbeta1 aporeceptor suppressed KCND3 expression, with both effects exhibiting ligand-dependent amplification upon T3 stimulation. Deletion of the KCND3 5'-flanking region localized the suppressible promoter sequence for TRbeta1 to within -293 bp and the activating promoter sequence for TRalpha1 to within -2335 to -1654 bp of the transcription start site. Disruption of putative TR binding sites by mutagenesis abolished the TRalpha1- (G-1651T) and TRbeta1- (G-73T) mediated effects, indicating that TRalpha1 and TRbeta1 response elements map to different regions of the KCND3 promoter. Thus, I(to) is modulated by diverse T3-dependent regulation of underlying gene transcription. TRalpha1 and TRbeta1 exhibit distinct effects on KCND3 transactivation with TRalpha1 enhancing and TRbeta1 suppressing KCND3 transcription.

The TRbeta1 is essential in mediating T3 action on Akt pathway in human pancreatic insulinoma cells.

Thyroid hormone action, widely recognized on cell proliferation and metabolism, has recently been related to the phosphoinositide 3 kinase (PI3K), an upstream regulator of the Akt kinase and the involvement of the thyroid hormone receptor beta1 has been hypothesized. The serine-threonine kinase Akt can regulate various substrates that drive cell mass proliferation and survival. Its action has also been characterized in pancreatic beta-cells. We previously demonstrated that Akt activity and its activation in the insulinoma cell line hCM could be considered a specific target of the non-genomic action of T3. In this study we analyzed the molecular pathways involved in the regulation of cell proliferation, survival, size, and protein synthesis by T3 in a stable TRbeta1 interfered insulinoma cell line, derived from the hCM, and evidenced a strong regulation of both physiological and molecular events by T3 mediated by the thyroid hormone receptor beta1. We showed that the thyroid receptor beta1 mediates the T3 regulation of the cdk4.cyc D1.p21(CIP1).p27(KIP1) complex formation and activity. In addition TRbeta1 is essential for the T3 upregulation of the Akt targets beta-catenin, p70S6K, and for the phosphorylation of Bad and mTOR. We demonstrated that the beta1 receptor mediates the T3 upregulation of protein synthesis and cell size, together with the cell proliferation and survival, playing a crucial role in the T3 regulation of the PI3K/Akt pathway.

Thyroid hormone receptor subtype specificity for hormone-dependent neurogenesis in Xenopus laevis.

Thyroid hormone (T(3)) influences cell proliferation, death and differentiation during development of the central nervous system (CNS). Hormone action is mediated by T(3) receptors (TR) of which there are two subtypes, TRalpha and TRbeta. Specific roles for TR subtypes in CNS development are poorly understood. We analyzed involvement of TRalpha and TRbeta in neural cell proliferation during metamorphosis of Xenopus laevis. Cell proliferation in the ventricular/subventricular neurogenic zones of the tadpole brain increased dramatically during metamorphosis. This increase was dependent on T(3) until mid-prometamorphosis, after which cell proliferation decreased and became refractory to T(3). Using double labeling fluorescent histochemistry with confocal microscopy we found TRalpha expressed throughout the tadpole brain, with strongest expression in proliferating cells. By contrast, TRbeta was expressed predominantly outside of neurogenic zones. To corroborate the histochemical results we transfected living tadpole brain with a Xenopus TRbeta promoter-EGFP plasmid and found that most EGFP expressing cells were not dividing. Lastly, treatment with the TRalpha selective agonist CO23 increased brain cell proliferation; whereas, treatment with the TRbeta-selective agonists GC1 or GC24 did not. Our findings support the view that T(3) acts to induce cell proliferation in the tadpole brain predominantly, if not exclusively, via TRalpha.

Distinct modulatory roles for thyroid hormone receptors TRalpha and TRbeta in SREBP1-activated ABCD2 expression.

Adrenoleukodystrophy-related protein, a peroxisomal ABC transporter encoded by ABCD2, displays functional redundancy with the disease-associated X-linked adrenoleukodystrophy protein, making pharmacological induction of ABCD2 a potentially attractive therapeutic approach. Sterol regulatory element (SRE)-binding proteins (SREBPs) induce ABCD2 through an SRE overlapping with a direct repeat (DR-4) element. Here we show that thyroid hormone (T(3)) receptor (TR)alpha and TRbeta bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TRbeta, but not TRalpha, represses ABCD2 induction independently of DNA binding. However, activation by TRalpha and derepression of TRbeta are T(3)-dependent and require intact SRE/DR-4 motifs. Electrophoretic mobility shift assays with nuclear extracts support a direct interaction of TR and SREBP1 at the SRE/DR-4. In the liver, Abcd2 expression is high in young mice (with high T(3) and TRalpha levels) but downregulated in adults (with low T(3) and TRalpha but elevated TRbeta levels). This temporal repression of Abcd2 is blunted in TRbeta-deficient mice, and the response to manipulated T(3) states is abrogated in TRalpha-deficient mice. These findings show that TRalpha and TRbeta differentially modulate SREBP1-activated ABCD2 expression at overlapping SRE/DR-4 elements, suggesting a novel mode of cross-talk between TR and SREBP in gene regulation.