Evolution of thyroid hormone signaling in animals: Non-genomic and genomic modes of action.

Much research has focused on vertebrate thyroid hormone (TH) synthesis and their function in development and metabolism. While important differences in TH synthesis and signaling exist, comparative studies between vertebrates fail to explain the evolutionary origins of this important regulatory axis. For that, one needs to make sense out of the diverse TH effects which have been described in invertebrate phyla but for which a mechanistic understanding is largely missing. Almost every major group of non-vertebrate animals possesses the capability to synthesize and metabolize thyroid hormones and there is evidence for a nuclear thyroid hormone receptor mediated mechanism in the bilateria, especially in molluscs, echinoderms, cephalochordates and ascidians. Still, genomic pathways cannot fully explain many observed effects of thyroid hormones in groups such as cnidarians, molluscs, and echinoderms and it is therefore possible that TH may signal via other mechanisms, such as non-genomic signaling systems via membrane bound or cytoplasmic receptors. Here we provide a brief review of TH actions in selected invertebrate species and discuss the hypothesis that non-genomic TH action may have played a critical role in TH signaling throughout animal evolution.

Cardiac hypertrophy and thyroid hormone signaling.

Thyroid hormone exerts a large number of influences on the cardiovascular system. Increased thyroid hormone action increases the force and speed of systolic contraction and the speed of diastolic relaxation and these are largely beneficial effects. Furthermore, thyroid hormone has marked electrophysiological effects increasing heart rate and the propensity for atrial fibrillation and these effects are largely mal-adaptive. In addition, thyroid hormone markedly increases cardiac angiogenesis and decreases vascular tone. These multiple thyroid hormone effects are largely mediated by the action of nuclear based thyroid hormone receptors (TR) the thyroid hormone receptor alpha and beta. TRalpha is the predominant isoform in the heart. Rapid nongenomic thyroid hormone effects also occur, which can be clearly demonstrated in ex-vivo experiments. Some of the most marked thyroid hormone effects in cardiac myocytes involve influences on calcium flux, with thyroid hormone promoting expression of the gene encoding the calcium pump of the sarcoplasmic reticulum (SERCa2). In contrast, in hypothyroid animals phospholamban levels, which inhibit the SERCa2 pump, are increased. In addition, marked effects are exerted on the calcium channel of the sarcoplasmic reticulum the ryanodine channel. Related to myofibrillar proteins, myosin heavy chain alpha is increased by T3 and MHC beta is decreased. Complex and interesting interactions occur between cardiac hypertrophy induced by excess thyroid hormone action and cardiac hypertrophy occurring with heart failure. The thyroid hormone mediated cardiac hypertrophy in its initial phases presents a physiological hypertrophy with increases in SERCa2 levels and decreased expression of MHC beta. In contrast, pressure overload induced heart failure leads to a "pathological" cardiac hypertrophy which is largely mediated by activation of the calcineurin system and the MAPkinases signaling system. Recent evidence indicates that heart failure can lead to a downregulation of the thyroid hormone signaling system in the heart. In the failing heart, decreases of thyroid hormone receptor levels occur. In addition, serum levels of T4 and T3 are decreased with heart failure in the frame of the non-thyroidal illness syndrome. The decrease in T3 serves as an indicator for a bad prognosis in the heart failure patient being linked to increased mortality. In animal models, it can be shown that in pressure overload-induced cardiac hypertrophy a decrease of thyroid hormone receptor levels occurs. Cardiac function can be improved by increasing expression of thyroid hormone receptors mediated by adeno-associated virus based gene transfer. The failing heart may develop a "hypothyroid" status contributing to diminished cardiac contractile function.

Molecular characterization and sex-related seasonal expression of thyroid receptor subtypes in goldfish.

The thyroid hormones, acting through the nuclear thyroid receptors (TRs), play important roles in the growth and development of vertebrates. The present study investigated the molecular structure and season-related expression of the TR isoforms in the male and female goldfish pituitary, brain, liver, gonads, gut, heart, and muscle. Based on sequence alignment with other species, the results demonstrate the presence of: (1) a TRalpha (TRalpha-1) consisting of 1496 nucleotides encoding a 466 amino acid protein, (2) a novel splice variant of TRalpha (TRalpha-2) containing an out-of-frame deletion of 246 nucleotides in the ligand-binding domain consisting of 1251 nucleotides encoding a 378 amino acid protein, (3) a novel transcript resembling TRalpha, except for non-homology in the hinge region and a premature stop codon prior to the ligand-binding domain (TRalpha-truncated; 1418 nucleotides, 206 amino acid protein), and (4) TRbeta consisting of 1823 nucleotides encoding a 395 amino acid protein. The findings provide the first demonstration of the presence of a truncated TR isoform in non-mammalian vertebrates. In goldfish, the expression patterns for all TRs subtypes were found to be remarkably similar in both male and female, changing significantly before and during reproductive season. The results provide a frame work for better understanding of the functional significance of novel TR forms and TR subtypes in fish and other vertebrates.

Hormone-dependent repression of the E2F-1 gene by thyroid hormone receptors.

Thyroid hormone induces differentiation of many different tissues in mammals, birds, and amphibians. The different tissues all differentiate from proliferating precursor cells, and the normal cell cycle is suspended while cells undergo differentiation. We have investigated how thyroid hormone affects the expression of the E2F-1 protein, a key transcription factor that controls G1- to S-phase transition. We show that during thyroid hormone-induced differentiation of embryonic carcinoma cells and of oligodendrocyte precursor cells, the levels of E2F-1 mRNA and E2F-1 protein decrease. This is caused by the thyroid hormone receptor (TR) regulating the transcription of the E2F-1 gene. The TR binds directly to a negative thyroid hormone response element, called the Z-element, in the E2F-1 promoter. When bound, the TR activates transcription in the absence of ligand but represses transcription in the presence of ligand. In addition, liganded TR represses transcription of the S-phase-specific DNA polymerase alpha, thymidine kinase, and dihydropholate reductase genes. These results suggest that thyroid hormone-induced withdrawal from the cell cycle takes place through the repression of S-phase genes. We suggest that this is an initial and crucial step in thyroid hormone-induced differentiation of precursor cells.

Glucocorticoid and thyroid hormone receptors in mitochondria of animal cells.

This article concerns the localization of glucocorticoid and thyroid hormone receptors in mitochondria of animal cells. The receptors are discussed in terms of their potential role in the regulation of mitochondrial transcription and energy production by the oxidative phosphorylation pathway, realized both by nuclear-encoded and mitochondrially encoded enzymes. A brief survey of the role of glucocorticoid and thyroid hormones on energy metabolism is presented, followed by a description of the molecular mode of action of these hormones and of the central role of the receptors in regulation of transcription. Subsequently, the structure and characteristics of glucocorticoid and thyroid hormone receptors are described, followed by a section on the effects of glucocorticoid and thyroid hormones on the transcription of mitochondrial and nuclear genes encoding subunits of OXPHOS and by an introduction to the mitochondrial genome and its transcription. A comprehensive description of the data demonstrates the localization of glucocorticoid and thyroid hormone receptors in mitochondria as well as the detection of potential hormone response elements that bind to these receptors. This leads to the conclusion that the receptors potentially play a role in the regulation of transcription of mitochondrial genes. The in organello mitochondrial system, which is capable of sustaining transcription in the absence of nuclear participation, is presented, responding to T3 with increased transcription rates, and the central role of a thyroid receptor isoform in the transcription effect is emphasized. Lastly, possible ways of coordinating nuclear and mitochondrial gene transcription in response to glucocorticoid and thyroid hormones are discussed, the hormones acting directly on the genes of the two compartments by way of common hormone response elements and indirectly on mitochondrial genes by stimulation of nuclear-encoded transcription factors.

Downregulation of thyroid hormone receptor subtype mRNA levels by retinoic acid in cultured cardiomyocytes.

The thyroid hormone receptors (TR) and the retinoic acid receptors share a high degree of homology and their signaling pathways interplay. Thyroid hormone (T3) is known to be associated with various pathological heart conditions. Retinoids are known to ameliorate symptoms in hyperthyroid patients. The aim of this study was to investigate if retinoic acid (RA) can have any effects on TR in cardiac cells and thus play a role in heart disease. Confluent AT-1 cardiomyocytes were treated with RA, T3 depleted medium and DITPA (a cardiotonic T3 analogue) for 48 hours. Solution hybridization for the determination of mRNA for TR alpha 1, alpha 1, beta 1 and beta 2 was performed. RA, T3 and DITPA significantly downregulated the alpha 1, beta 1 and beta 2. The T3 depleted medium did not affect the TR subtypes. The specificity of the solution hybridization method was tested by an RNase protection assay. In conclusion, RA downregulates TR in a similar way as T3 in cardiac cells, indicating a role for RA in thyroid associated heart disease.

Differential gene expression of thyroid hormone receptor alpha and beta in fish development.

To understand the mechanisms of flounder metamorphosis, which is controlled by thyroid hormone, gene expression of the thyroid hormone receptors (TR alpha A, TR alpha B, TR beta 1, and TR beta 2) was studied in developing flounder larvae, using quantitative reverse-transcriptase polymerase chain reaction and in situ hybridization. TR gene transcripts were found at very low levels in fertilized flounder eggs. Substantial amounts of TR mRNAs were present in premetamorphic larvae, except for TR alpha B mRNA, which was low throughout larval development. TR alpha A gene transcripts increased rapidly in metamorphic climax and decreased rapidly postclimax. The expression level of TR beta s increased in climax, reached its peak postclimax, and remained high in metamorphosed juveniles. In situ hybridization confirmed the decrease in TR alpha transcripts in most tissues postclimax and further revealed the ubiquitous expression of TR genes and distinct tissue specificity of alpha and beta subtypes in the overall fish body. These results suggest that thyroid hormone exerts effects directly on each tissue during fish metamorphosis and that gene expression of TR subtypes is differentially regulated both temporally and regionally. Thus, the results suggest that the development of each tissue of the flounder by thyroid hormone is further controlled at the receptor level by the differential expression of TRs.

A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily.

The steroid/hormone nuclear receptor superfamily comprises several subfamilies of receptors that interact with overlapping DNA sequences and/or related ligands. The thyroid/retinoid hormone receptor subfamily has recently attracted much interest because of the complex network of its receptor interactions. The retinoid X receptors (RXRs), for instance, play a very central role in this subfamily, forming heterodimers with several receptors. Here we describe a novel member of this subfamily that interacts with RXR. Using a v-erbA probe, we obtained a cDNA which encodes a novel 445-amino-acid protein, RLD-1, that contains the characteristic domains of nuclear receptors. Northern (RNA) blot analysis showed that in mature rats, the receptor is highly expressed in spleen, pituitary, lung, liver, and fat. In addition, weaker expression is observed in several other tissues. Amino acid sequence alignment and DNA-binding data revealed that the DNA-binding domain of the new receptor is related to that of the thyroid/retinoid subgroup of nuclear receptors. RLD-1 preferentially binds as a heterodimer with RXR to a direct repeat of the half-site sequence 5'-G/AGGTCA-3', separated by four nucleotides (DR-4). Surprisingly, this binding is dependent to a high degree on the nature of the spacing nucleotides. None of the known nuclear receptor ligands activated RLD-1. In contrast, a DR-4-dependent constitutive transcriptional activation of a chloramphenicol acetyltransferase reporter gene by the RLD-1/RXR alpha heterodimer was observed. Our data suggest a highly specific role for this novel receptor within the network of gene regulation by the thyroid/retinoid receptor subfamily.

Expression of nuclear hormone receptors within the rat hippocampus: identification of novel orphan receptors.

Within the hippocampus, stimulus-transcriptional coupling plays an important role in post-seizure neuronal adaptation, post-ischemic cell death and the induction of long-term potentiation. To identify additional mediators of hippocampal transcriptional responses a targeted approach was developed and used to characterize the spectrum of nuclear hormone receptors expressed within this brain region. cDNAs encoding the DNA-binding domains of six different members of the nuclear hormone receptor superfamily were isolated. A majority were identical or closely related to receptors known to be expressed within the hippocampus. Two additional isolates, HZF-2 and HZF-3, encode the DNA-binding domain of novel members of the nuclear hormone receptor superfamily.

Spatial and temporal expression of alpha- and beta-thyroid hormone receptor mRNAs, including the beta 2-subtype, in the developing mammalian nervous system.

Thyroid hormone exerts profound effects on the developing mammalian brain, and its deficiency can lead to severe mental retardation and motor abnormalities. To identify specific anatomic targets of thyroid hormone action in the developing mammalian nervous system, we examined thyroid hormone receptor gene expression by hybridization histochemistry on serial adjacent sections from 12 stages of the developing rat nervous system. 35S-labeled cRNA probes were generated from divergent sequences of rat alpha 1-, alpha 2-, beta 1-, and beta 2-thyroid hormone receptor and related cDNAs. We found that alpha- and beta-thyroid hormone receptor genes have distinct patterns of spatiotemporal expression in the embryonic and postnatal rat nervous system. alpha 1- and alpha 2-mRNAs were widely expressed in similar patterns; highest levels were found in the fetal neocortical plate, site of cortical neuronal differentiation. In contrast, beta 1-transcripts were restricted in distribution, with prominent expression in zones of neuroblast proliferation such as the germinal trigone and the cortical ventricular layer. Surprisingly, the "pituitary-specific" beta 2-transcript was detected in the developing hippocampus and striatum. Our results suggest that alpha- and beta-thyroid hormone receptors may play distinct functional roles during development of the mammalian nervous system.

Differential modes of activation define orphan subclasses within the steroid/thyroid receptor superfamily.

We report that three orphan receptors, hERR1, hERR2, and hTR2, members of the steroid/thyroid receptor (SR/TR) superfamily, can be activated by different ligand-independent pathways. hERR1 and hERR2 exhibit constitutive activity in the absence of exogenously added ligands. Furthermore, this constitutive activity is localized in the carboxy terminal domain of both receptors and can be transferred to other members of this superfamily using domain switch strategies. In addition, we show that hERR1 can remain constitutively active in the less evolved eukaryotic cell Saccharomyces cerevisiae. In contrast, hTR2 is not constitutively active. However, a chimera of hTR2 can be activated in a ligand-independent manner through a signal transduction pathway initiated at the cell membrane by the neurotransmitter dopamine. Like hERR1 and hERR2, hTR2 is ligand-independently activated through its carboxy terminal domain. Together, these results suggest the existence of emerging subgroups within the SR/TR superfamily that can regulate gene expression through different modes of activation.

The roles of three forms of human thyroid hormone receptor in gene regulation.

We have expressed three forms of human thyroid hormone receptor (hTR alpha 1, alpha 2, and beta) in cultured cells by transient transfection. hTR alpha 1 and beta transfected cells showed increased triiodothyronine (T3) binding capacity, but hTR alpha 2 transfected cells did not. When hTR alpha 1 or beta was cotransfected with pUrGH(S), in which a portion of the rat GH 5' flanking region (-236/-147) was ligated into the CAT reporter plasmid (pUTKAT1), T3 increased CAT gene expression. When hTR alpha 2 was cotransfected with pUrGH(S), T3 did not alter CAT gene expression. When hTR alpha 1 or beta was cotransfected with pUrGH(O), in which a synthetic oligonucleotide representing the TRE from the rat GH 5' flanking region (-189/-160) was substituted for the natural enhancer in pUTKAT1, T3 increased CAT gene expression. When hTR alpha 1 and beta were cotransfected with pUrGH(O), induction by T3 was increased. When hTR alpha 2 was cotransfected with hTR alpha 1 or beta, induction by T3 was decreased. These results indicate that hTR alpha 1 and beta function as native TR, that hTR alpha 1 and beta can recognize the same TRE, that hTR alpha 1 and beta can function additively, and that hTR alpha 2 can inhibit the action of hTR alpha 1 and beta.

Thyroid hormone receptor expression in the "sick euthyroid" syndrome.

To explore the hypothesis that alteration of T3 receptor expression may be an important mechanism controlling the tissue effects of thyroid hormones in the "sick euthyroid" syndrome, specific triiodothyronine (T3) receptor mRNAs were measured in tissues from normal subjects and from patients with liver disease, chronic renal failure, or with multiple organ failure on an intensive care unit (ICU). In all patient groups circulating free thyroxine and free T3 were reduced, while thyroid stimulating hormone remained normal. In patients with liver or renal disease, there were significant increases in levels of both alpha and beta T3 receptor mRNAs in peripheral mononuclear cells (PMNCs); in ICU patients there was a significant increase in beta mRNA. In patients with liver disease increases in T3 receptor mRNAs were not confined to PMNCs but were also found in liver biopsy specimens when levels were compared with those in normal donor liver. After liver transplantation, receptor mRNAs in PMNCs were similar to those in controls; likewise beta mRNA was similar in liver tissue to normal liver. There was, however, persistent elevation in alpha receptor mRNA. Increases in T3 receptor expression in non-thyroidal illness may be responsible for the maintenance of euthyroidism in the face of reduced levels of circulating thyroid hormones.