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.     

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.     

The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells

The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.     

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.     

Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver

Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-alpha encoding TRalpha1 and TRalpha2 and their respective Delta isoforms, and c-erbA-beta which encodes TRbeta1, TRbeta2 and TRbeta3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TRbeta1 isoform in rat liver. We were therefore also interested to see whether TRalpha1 and TRalpha2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TRalpha1 and TRalpha2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TRalpha1 and TRalpha2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TRalpha1 is broader than that of TRbeta1. Moreover, the expression of the TRalpha2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TRalpha1 protein.From our data it appears that both the TRalpha1 and TRalpha2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.     

Effects of thyroid hormone analogs on lipid metabolism and thermogenesis

Thyroid hormone affects in a myriad of biological processes such as development, growth, and metabolic control. Triiodothyronine (T3) is the biologically active form of thyroid hormone that acts through nuclear receptors, TRalpha and TRbeta, regulating gene expression. Given that the distribution of these receptors is heterogeneous amongst the different tissues, it is not surprising that some physiological effects of T3 are isoform specific. For example, while TRalpha is the dominant receptor in the brain and skeletal system and mediates most of the synergism between T3 and the sympathetic signaling pathway in the heart, TRbeta is abundant in liver and is probably the isoform that mediates most of the T3 effects on lipid metabolism. Thus, it makes sense to develop compounds that selectively act on either one of the TRs, allowing for the activation of specific T3-dependent pathways. This article reviews the recent progress made in this area, focusing on the physiological effects of compounds that lower serum cholesterol and decrease fat mass, as they spare skeletal muscle and bone masses, as well as the heart. The available studies indicate that achieving selective activation of different TR-mediated pathways is a promising strategy for treating lipid disorders and obesity.