Hyper and hypothyroidism change the expression and diurnal variation of thyroid hormone receptor isoforms in rat liver without major changes in their zonal distribution

We investigated the effect of hypothyroidism or hyperthyroidism on mRNA and protein expression, diurnal variation and zonal distribution of thyroid hormone receptor (TR) isoforms TRalpha1, TRalpha2 and TRbeta1 in rat liver. Hypothyroidism results in increased isoform mRNA and protein expression whereas hyperthyroidism shows a decreased TRalpha1 and TRalpha2 mRNA and protein expression. During hyperthyroidism no change is seen in TRbeta1 mRNA, but TRbeta1 protein is upregulated in the light period and downregulated in the dark period. Diurnal changes (measured at 13:30 and 19:30 h) in the TR isoform proteins are abolished in hypothyroidism and hyperthyroidism, with the exception of a reversal in diurnal changes of TRbeta1 in hyperthyroidism. Zonal distribution of the isoforms is not affected by hypo- or hyperthyroidism. We therefore conclude that thyroid hormone influences both the levels and the diurnal expression of its receptor isoforms but not the zonal distribution.     

Thyroid hormone receptor isoform expression in livers of critically ill patients.

OBJECTIVE: The THRA gene encodes two isoforms of the thyroid hormone receptor (TR), TRalpha1 and TRalpha2. The ratio of these splice variants could have a marked influence on T3-regulated gene expression, especially during illness. DESIGN: We studied the expression of the isoforms TRbeta1, TRalpha1, and TRalpha2 and 5'-deiodinase in postmortem liver biopsies of 58 patients who were critically ill and died in the intensive care unit (ICU). All mRNA levels were determined using real-time PCR. MAIN OUTCOME: All ratios of the biopsies were higher than those found in three normal liver biopsies due to an increased TRalpha1 level. The TRalpha1/TRalpha2 ratio increased with age and severity of illness following the equation: TRalpha1/TRalpha2 ratio = - 1.854 + (0.0323 x age) + (0.0431 x Therapeutic Intervention Scoring System score) indicating that 28% of the changed TRalpha1/TRalpha2 ratio can be predicted by these clinical variables. There was no effect of randomization to intensive insulin therapy or glucocorticoid or thyroid hormone treatment on the TRalpha1/TRalpha2 ratio or TRbeta1. Furthermore, no relation was seen between the expression levels of the 5'-deiodinase mRNA and TR isoforms or the triiodothyronine T3 levels. CONCLUSION: It appears that in critically ill patients the ratio of TRalpha1/TRalpha2 expression increases with age and severity of illness, possibly indicating a mechanism to enhance sensitivity to T3 in the oldest and sickest patients.     

Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive posttranscriptional regulation

The perinatal changes in the pattern of expression of the thyroid hormone receptor (TR) isoforms TRalpha (1) TRalpha (2), TRbeta (1), and TRbeta (2) were investigated using in situ hybridization and immunohistochemistry, and RT-PCR and western blotting as visualization and quantification techniques respectively. In liver, lung, and kidney, TRalpha mRNA was expressed in the stromal and TRbeta mRNA in the parenchymal component of the tissues. When compared with liver, TRalpha mRNA concentrations were tenfold higher in lung, kidney, and intestine, and 100-fold higher in brain, with TRalpha (2) mRNA concentrations exceeding those of TRalpha (1) 5-to 10-fold. Tissue TRbeta (1) mRNA concentrations were similar in liver, lung, and brain, and 3- to 5-fold higher in kidney and intestine. None of the TRbeta (2) mRNA could be detected outside the pituitary. Tissue TRalpha (2) and TRbeta (1) protein levels reached adult levels at 5 days before birth, whereas TRalpha (1) protein peaked after birth. Because of the distinct time-course of thyroid hormone-binding receptors TRalpha (1) and TRbeta (1), we speculate that an initiating, TRbeta (1)-mediated signaling from the parenchyma is followed by a TRalpha (1)-mediated response in the stroma. When compared with organs with a complementary parenchymal-stromal expression pattern, organs with extensive cellular co-expression of TRalpha and TRbeta (brain and intestinal epithelium) were characterized by a very low TRalpha protein: mRNA ratio, implying a low translational efficiency of TR mRNA or a high turnover of TR protein. The data indicate that the TR-dependent regulatory cascades are controlled differently in organs with a complementary tissue expression pattern and in those with cellular co-expression of the TRalpha and TRbeta genes.     

Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart

Nuclear thyroid hormone (T3) receptors (TR) play a critical role in mediating the effects of T3 on development, differentiation and normal physiology of many organs. The heart is a major target organ of T3, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRalpha1, TRalpha2 and TRbeta1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRalpha1, TRalpha2 and TRbeta1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRbeta1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRalpha1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRalpha1 and TRbeta1 isoforms are co-expressed. We also found the heterogeneous expression of the TRalpha1, TRalpha2 and TRbeta1 isoforms in the developing mouse heart, which, in the case of the TRbeta1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRbeta1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRalpha1 and TRbeta1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.     

Differential expression of thyroid hormone receptor isoforms is strikingly related to cardiac and skeletal muscle phenotype during postnatal development.

The genomic actions of thyroid hormones (THs) are mediated by receptors (TRs) that are encoded by two protooncogenes, c-erbA-alpha and c-erbA-beta. The precise functions of the TR isoforms are unclear and this study focuses on the potential roles of the TRalpha and TRbeta isoforms in mammalian striated muscles postnatally. The porcine TRalpha1, TRalpha2 and TRbeta1 cDNAs were first cloned, sequenced and characterised by Northern blotting. A quantitative analysis of TR isoform expression was then undertaken, using RNase protection analysis with novel riboprobes designed to detect relative expression levels of TRalpha1, TRalpha2, TRbeta1 and TRbeta2, in functionally distinct muscles from 7-week-old pigs kept under controlled conditions of nutrition and thermal environment. We found a striking muscle-specific pattern of TRalpha isoform distribution: in heart the mRNA level of TRalpha2 (non-TH binding) was markedly greater (P<0.01) than that of TRalpha1 (TH binding); in longissimus dorsi the opposite pattern of expression occurred (TRalpha1>TRalpha2, P<0.001); in soleus, diaphragm and rhomboideus there were no differences between the two isoforms. The overall abundance of TRbeta was very much lower than that of TRalpha, and TRbeta1 was expressed at a higher level than TRbeta2 in all muscles. Together with recent data from TR gene inactivation studies and the established role of TH in determining myosin heavy chain isoform expression and muscle phenotype, these results suggest a role for differential expression of TR isoforms in acquisition and maintenance of optimal cardiac and skeletal muscle function.     

Increased sensitivity to thyroid hormone in mice with complete deficiency of thyroid hormone receptor alpha

Only three of the four thyroid hormone receptor (TR) isoforms, alpha1, beta1, and beta2, bind thyroid hormone (TH) and are considered to be true TRs. TRalpha2 binds to TH response elements on DNA, but its role in vivo is still unknown. We produced mice completely deficient in TRalpha (TRalpha(o/o)) that maintain normal serum thyroid-stimulating hormone (TSH) concentration despite low serum thyroxine (T(4)), suggesting increased sensitivity to TH. We therefore examined the effects of TH (L-3,3',5-triiodothyronine, L-T3) given to TH-deprived and to intact TRalpha(o/o) mice. Controls were wild-type (WT) mice of the same strain and mice resistant to TH due to deficiency in TRbeta (TRbeta(-/-)). In liver, T3 produced significantly greater responses in TRalpha(o/o) and smaller responses in TRbeta(-/-) as compared with WT mice. In contrast, cardiac responses to L-T3 were absent or reduced in TRalpha(o/o), whereas they were similar in WT and TRbeta(-/-) mice, supporting the notion that TRalpha1 is the dominant TH-dependent TR isoform in heart. 5-Triiodothyronine (L-T3) given to intact mice produced a greater suppression of serum T(4) in TRalpha(o/o) than it did in WT mice and reduced by a greater amount the TSH response to TSH-releasing hormone. This is an in vivo demonstration that a TR deficiency can enhance sensitivity to TH. This effect is likely due to the abrogation of the constitutive "silencing" effect of TRalpha2 in tissues expressing the TRbeta isoforms.     

Ontogenetic pattern of thyroid hormone receptor expression in the human testis

We studied the spatiotemporal distribution of thyroid hormone nuclear receptors (TRs) alpha1 and alpha2 and beta messenger RNA (mRNA) levels in normal human testicular tissue during development and in adulthood. Nonpathological specimens from five aborted fetuses (17 and 23 weeks of gestation, three and two cases, respectively) and from four patients undergoing orchiectomy (18 months old and 38-, 42-, and 52-yr-old, respectively) were analyzed by Northern blot, semiquantitative RT-PCR amplification using DNA sequences or specifically designed primers for the TR isoforms, and in situ hybridization. By using PCR amplification, we found that TRalpha1 and TRalpha2 are both expressed at different levels in fetal and adult testis. At all ages TRalpha2 is found at higher levels. Northern analysis showed hybridization signals corresponding to the expression of TRalpha2 and TRalpha in a ratio that increased from 2.6 at 17 weeks of gestation to 12.0 in adulthood. In fact, the expression of TRalpha1 dramatically decreased throughout development, being faintly detectable in the adult testis. Expression of TRbeta was not detected at any age studied. This finding was further confirmed by PCR, which did not amplify TRbeta either in fetal or in adult testis mRNAs. In situ hybridization studies showed the absence of TRbeta and that TRalpha1 and TRalpha2 colocalized in Sertoli cells of prepubertal testis, whereas germ and interstitial cells appeared devoid of TR mRNA signals. From these results it can be concluded that the human testis exclusively expresses TRalpha, which is localized in Sertoli cells, TRbeta being always undetectable. Fetal and prepubertal ages represent the period of maximal expression of TRalpha1 and TRalpha2. The alpha2/alpha1 ratio rises dramatically after development. These results confirm a critical window for the action of thyroid hormone in human testis, in the period of maximal expression of T3 binding isoform TRalpha1, and may account for the macroorchidism without virilization occurring when hyposecretion of thyroid hormones occurs before puberty.     

Thyroid hormone receptors: lessons from knockout and knock-in mutant mice.

The genes encoding thyroid hormone receptor alpha and beta (TRalpha and TRbeta) encode four thyroid hormone receptors and four variant isoforms with antagonistic properties. Because of this complexity, numerous models of TR mutation have been developed to understand the functions of specific receptors. In total, 13 mutant strains are now available. Phenotype analysis has shown that the two genes serve distinct functions: TRalpha is crucial for postnatal development and cardiac function, whereas TRbeta mainly controls inner ear and retina development, liver metabolism and thyroid hormone levels. These mouse mutant strains also provide us with the unique opportunity to address the respective contribution of each receptor isoform and isotype in vivo and highlight the in vivo importance of the ligand-independent function of the TR gene products.     

A targeted dominant negative mutation of the thyroid hormone α1 receptor causes increased mortality, infertility, and dwarfism in mice

Mutations in the thyroid hormone receptor beta (TRbeta) gene result in resistance to thyroid hormone. However, it is unknown whether mutations in the TRalpha gene could lead to a similar disease. To address this question, we prepared mutant mice by targeting mutant thyroid hormone receptor kindred PV (PV) mutation to the TRalpha gene locus by means of homologous recombination (TRalpha1PV mice). The PV mutation was derived from a patient with severe resistance to thyroid hormone that has a frameshift of the C-terminal 14 aa of TRbeta1. We knocked in the same PV mutation to the corresponding TRalpha gene locus to compare the phenotypes of TRalpha1(PV/+) mice with those of TRbeta(PV/+) mice. TRalpha1(PV/+) mice were viable, indicating that the mutation of the TRalpha gene is not embryonic lethal. In drastic contrast to the TRbeta(PV/+) mice, which do not exhibit a growth abnormality, TRalpha1(PV/+) mice were dwarfs. These dwarfs exhibited increased mortality and reduced fertility. In contrast to TRbeta(PV/+) mice, which have a hyperactive thyroid, TRalpha1(PV/+) mice exhibited mild thyroid failure. The in vivo pattern of abnormal regulation of T3 target genes in TRalpha1(PV/+) mice was unique from those of TRbeta(PV/+) mice. The distinct phenotypes exhibited by TRalpha1(PV/+) and TRbeta(PV/+) mice indicate that the in vivo functions of TR mutants are isoform-dependent. The TRalpha1(PV/+) mice may be used as a tool to uncover human diseases associated with mutations in the TRalpha gene and, furthermore, to understand the molecular mechanisms by which TR isoforms exert their biological activities.     

Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components

OBJECTIVE: In view of their different actions on thyroid hormone receptor (TR) isoforms we set out to investigate whether amiodarone (AM) and dronedarone (Dron) have different and/or component-specific effects on cardiac gene expression. DESIGN: Rats were treated with AM or Dron and the expression of TRalpha 1, TRalpha 2, TRbeta 1 and several tri-iodothyronine (T3)-regulated genes was studied in different parts of the heart, namely the right atrium (RA), left ventricular wall (LVW) and apex. METHODS: Rats were treated for 14 days with 100 mg/kg body weight AM or Dron. The expression of TRalpha 1, TRalpha 2, TRbeta 1 and T3-regulated genes was studied using real-time PCR and non-radioactive in situ hybridisation. RESULTS: AM and Dron affected TR expression in the RA similarly by decreasing TRalpha 1 and beta 1 expression by about 50%. In the LVW, AM and Dron decreased TRbeta 1 and, interestingly, AM increased TRalpha 1. In the apex, AM also increased TRalpha 2. The changes seen in T3-dependent gene expression are reminiscent of foetal reprogramming. CONCLUSION: Taken together, our results indicate that AM and Dron have similar effects on the expression of TR isoforms in the RA, which could partly contribute to their ability to decrease heart rate. On the other hand, the more profound effect of AM appears on TR- and T3-dependent gene expression in the left ventricle suggests foetal reprogramming.