Distinct thyroid hormone‐dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH‐dependent period of the cochlea

Analyzing the thyroid hromone (TH)‐dependent period of the inner ear, we observed that the presence of triiodothyronine (T3) between postnatal day 3 (P3) and P12 is sufficient for functional maturation of the auditory system. Within this short time period, an unusual transient TH‐dependent expression of nonneuronal neurotrophin receptors (NT‐R) trkB and p75^NGFR was observed in correlation with neuronal and morphogenetic processes. The availability of thyroid hormone was revealed to be invariably correlated with (a) a transient expression of full‐length trkB in TRα1‐, TRα2‐ and TRβ1‐expressing hair cells concomitant to the segregation of afferent fibers and the synaptogenesis of efferent fibers; and (b) a transient expression of p75^NGFR in TRα1‐ and TRβ1‐expressing great epithelia ridge cells in direct spatiotemporal correlation with the appearance of apoptotic cells and morphogenetic maturation of the organ. For the first time, these data suggest a TH dependency of the expression of neurotrophin receptors in nonneuronal cells. A potential role of these peculiar neurotrophin receptor expression for the conversion of the biological function of TH on innervation patterning and morphogenesis during the critical TH‐dependent period of the inner ear may be considered. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 338–356, 1999     

Normal timing of oligodendrocyte development depends on thyroid hormone receptor alpha 1 (TRalpha1)

The timing of oligodendrocyte development is regulated by thyroid hormone (TH) in vitro and in vivo, but it is still uncertain which TH receptors mediate this regulation. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Here, we provide direct evidence for the involvement of the TRalpha1 receptor isoform in vivo, by showing that the number of oligodendrocytes in the postnatal day 7 (P7) and P14 optic nerve of TRalpha1-/- mice is decreased compared with normal. We demonstrate that TRalpha1 mediates the normal differentiation-promoting effect of TH on oligodendrocyte precursor cells (OPCs): unlike wild-type OPCs, postnatal TRalpha1-/- OPCs fail to stop dividing and differentiate in response to TH in culture. We also show that overexpression of TRalpha1 accelerates oligodendrocyte differentiation in culture, suggesting that the level of TRalpha1 expression is normally limiting for TH-dependent OPC differentiation. Finally, we provide evidence that the inhibitory isoforms of TRalpha are unlikely to play a part in the timing of OPC differentiation.     

Developmental expression analysis of thyroid hormone receptor isoforms reveals new insights into their essential functions in cardiac and skeletal muscles.

Nuclear thyroid hormone (TH) receptors (TR) play a critical role in mediating the diverse actions of TH in development, differentiation, and metabolism of most tissues, but the role of TR isoforms in muscle development and function is unclear. Therefore, we have undertaken a comprehensive expression analysis of TRalpha 1, TRbeta 1, TRbeta 2 (TH binding), and TRalpha 2 (non-TH binding) in functionally distinct porcine muscles during prenatal and postnatal development. Use of a novel and highly sensitive RNase protection assay revealed striking muscle-specific developmental profiles of all four TR isoform mRNAs in cardiac, longissimus, soleus, rhomboideus, and diaphragm. Distribution of TR isoforms varied markedly between muscles; TRalpha expression was considerably greater than TRbeta and there were significant differences in the ratios TRalpha 1:TRalpha 2, and TRbeta 1:TRbeta 2. Together with immunohistochemistry of myosin heavy chain isoforms and data on myogenesis and maturation of the TH axis, these findings provide new evidence that highlights central roles for 1) TRalpha isoforms in fetal myogenesis, 2) the ratio TRalpha 1:TRalpha 2 in determining cardiac and skeletal muscle phenotype and function; 3) TRbeta in maintaining a basal level of cellular response to TH throughout development and a specific maturational function around birth. These findings suggest that events disrupting normal developmental profiles of TR isoforms may impair optimal function of cardiac and skeletal muscles.     

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.     

Thyroid hormone receptor expression in the obligatory paedomorphic salamander Necturus maculosus

Amphibian metamorphosis is under the strict control of thyroid hormones (TH). These hormones induce metamorphosis by controlling gene expression through binding to thyroid hormone receptors (TRs). Necturus maculosus is considered to be an obligatory paedomorphic Amphibian since metamorphosis never occurs spontaneously and cannot be induced by pharmacological means. Since metamorphosis depends on the acquisition of response of tadpole tissues to thyroid hormone, we aimed to determine TR gene expression patterns in Necturus maculosus as well as the expression of two TH-related genes: Cytosolic Thyroid Hormone-Binding Protein (CTHBP)-M2-pyruvate kinase, a gene encoding a cytosolic TH binding protein and stromelysin 3, a direct TH target gene in Xenopus laevis. Tissue samples were obtained from specimens of Necturus maculosus. We performed in situ hybridization using non-cross-hybridizing RNA probes obtained from the cloned Necturus TRalpha and TRbeta genes. We found clear expression of Necturus TRalpha gene in several tissues including the central nervous system, epithelial cells of digestive and urinary organs, as well as myocardium and skeletal muscle. TRbeta was also expressed in the brain. In other tissues, hybridization signals were too low to draw reliable conclusions about their precise distribution. In addition, we observed that the expression of CTHBP and ST3 is largely distinct from that of TRs. The fact that we observed a clear expression of TRalpha and TRbeta which are evolutionary conserved, suggests that Necturus tissues express TRs. Our results thus indicate that, in contrast to previously held hypotheses, Necturus tissues are TH responsive.     

Role of thyroid hormone receptors in timing oligodendrocyte differentiation.

The timing of oligodendrocyte differentiation is thought to depend on both intracellular mechanisms and extracellular signals. Thyroid hormone (TH) helps control this timing both in vitro and in vivo, but it is still uncertain how it does so. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Previous studies suggested that TRbeta receptors may mediate the effect of TH on oligodendrocyte precursor cells (OPCs). Consistent with this possibility, we show here that overexpression of TRbeta1 promotes precocious oligodendrocyte differentiation, whereas expression of two dominant-negative forms of TRbeta1 greatly delays differentiation. Surprisingly, however, we find that postnatal TRbeta-/- mice have a normal number of oligodendrocytes in their optic nerves and that TRbeta-/- OPCs stop dividing and differentiate normally in response to TH in vitro. Moreover, we find that OPCs do not express TRbeta1 or TRbeta2 mRNAs, whereas they do express TRalpha1 and TRalpha2 mRNAs. These findings suggest that alpha receptors mediate the effect of TH on the timing of oligodendrocyte differentiation. We also show that TRalpha2 mRNA, which encodes a dominant-negative form of TRalpha, decreases as OPCs proliferate in vitro and in vivo. This decrease may help control when oligodendrocyte precursors differentiate.     

Thyroid hormone induces cardiac myocyte hypertrophy in a thyroid hormone receptor alpha1-specific manner that requires TAK1 and p38 mitogen-activated protein kinase

Alterations in TR [thyroid hormone (TH) receptor]1 isoform expression have been reported in models of both physiologic and pathologic cardiac hypertrophy as well as in patients with heart failure. In this report, we demonstrate that TH induces hypertrophy as a direct result of binding to the TRalpha1 isoform and, moreover, that overexpression of TRalpha1 alone is also associated with a hypertrophic phenotype, even in the absence of ligand. The mechanism of TH and TRalpha1-specific hypertrophy is novel for a nuclear hormone receptor and involves the transforming growth factor beta-activated kinase (TAK1) and p38. Mitigating TRalpha1 effects, both TRalpha2 and TRbeta1 attenuate TRalpha1-induced myocardial growth and gene expression by diminishing TAK1 and p38 activities, respectively. These findings refine our previous observations on TR expression in the hypertrophied and failing heart and suggest that manipulation of thyroid hormone signaling in an isoform-specific manner may be a relevant therapeutic target for altering the pathologic myocardial program.     

Cardiac glucose utilization in mice with mutated alpha- and beta-thyroid hormone receptors

Abnormal thyroid function is usually associated with altered cardiac function. Mutations in the thyroid hormone (TH)-binding region of the TH beta-receptor (TRbeta) that eliminate its TH-binding ability lead to the thyroid hormone resistance syndrome (RTH) in humans, which is characterized by high blood TH levels, goiter, hyperactivity, and tachycardia. Mice with "knock-in" mutations in the TH alpha-receptor (TRalpha) or TRbeta that remove their TH-binding ability have been developed, and those with the mutated TRbeta (TRbeta(PV/PV)) appear to provide a model for RTH. These two types of mutants show different effects on cerebral energy metabolism, e.g., negligible change in glucose utilization (CMR(Glc)) in TRbeta(PV/PV) mice and markedly reduced CMR(Glc), like that found in cretinous rats, in the mice (TRalpha(PV/+)) with the knock-in mutation of the TRalpha gene. Studies in knockout mice have indicated that the TRalpha may also influence heart rate. Because mutations in both receptor genes appear to affect some parameters of cardiac function and because cardiac functional activity and energy metabolism are linked, we measured heart glucose utilization (HMR(Glc)) in both the TRbeta(PV/PV) and TRalpha(PV/+) mutants. Compared with values in normal wild-type mice, HMR(Glc) was reduced (-77 to -95%) in TRalpha(PV/+) mutants and increased (87 to 340%) in TRbeta(PV/PV) mutants, the degree depending on the region of the heart. Thus the TRalpha(PV/+) and TRbeta(PV/PV) mutations lead, respectively, to opposite effects on energy metabolism in the heart that are consistent with the bradycardia seen in hypothyroidism and the tachycardia associated with hyperthyroidism and RTH.     

Thyroid hormone and cardiac function in mice deficient in thyroid hormone receptor-alpha or -beta: an echocardiograph study

We investigated the effect of thyroid hormone (TH) receptor (TR)alpha and -beta isoforms in TH action in the heart. Noninvasive echocardiographic measurements were made in mice homozygous for disruption of TRalpha (TRalpha(0/0)) or TRbeta (TRbeta(-/-)). Mice were studied at baseline, 4 wk after TH deprivation (using a low-iodine diet containing propylthiouracil), and after 4-wk treatment with TH. Baseline heart rates (HR) were similar in wild-type (WT) and TRalpha(0/0) mice but were greater in TRbeta(-/-) mice. With TH deprivation, HR decreased 49% in WT and 37% in TRbeta(-/-) mice and decreased only 5% in TRalpha(0/0) mice from baseline, whereas HR increased in all genotypes with TH treatment. Cardiac output (CO) and cardiac index (CI) in WT mice decreased (-31 and -32%, respectively) with TH deprivation and increased (+69 and +35%, respectively) with TH treatment. The effects of CO and CI were blunted with TH withdrawal in both TRalpha(0/0) (+8 and -2%, respectively) and TRbeta(-/-) mice (-17 and -18%, respectively). Treatment with TH resulted in a 64% increase in LV mass in WT and a 44% increase in TRalpha(0/0) mice but only a 6% increase in TRbeta(-/-) mice (ANOVA P < 0.05). Taken together, these data suggest that TRalpha and TRbeta play different roles in the physiology of TH action on the heart.     

Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development.

The biological activities of thyroid hormones are thought to be mediated by receptors generated by the TRalpha and TRbeta loci. The existence of several receptor isoforms suggests that different functions are mediated by specific isoforms and raises the possibility of functional redundancies. We have inactivated both TRalpha and TRbeta genes by homologous recombination in the mouse and compared the phenotypes of wild-type, and single and double mutant mice. We show by this method that the TRbeta receptors are the most potent regulators of the production of thyroid stimulating hormone (TSH). However, in the absence of TRbeta, the products of the TRalpha gene can fulfill this function as, in the absence of any receptors, TSH and thyroid hormone concentrations reach very high levels. We also show that TRbeta, in contrast to TRalpha, is dispensable for the normal development of bone and intestine. In bone, the disruption of both TRalpha and TRbeta genes does not modify the maturation delay observed in TRalpha -/- mice. In the ileum, the absence of any receptor results in a much more severe impairment than that observed in TRalpha -/- animals. We conclude that each of the two families of proteins mediate specific functions of triiodothyronin (T3), and that redundancy is only partial and concerns a limited number of functions.     

Thyroid hormone regulates tubulin expression in mammalian liver. Effects of deleting thyroid hormone receptor-alpha or -beta

Microtubules are made from polymers of alpha/beta dimers. We have observed in rat liver that, on the first day after birth, alpha-subunit is relatively high and beta-subunit low with respect to adult values. In the hypothyroid neonate, both subunits were found to be low, therefore indicating that thyroid hormone (TH) regulates these developmental changes. TH was also found to activate tubulin expression in adult liver, especially beta-subunit. To investigate the role of TH receptors (TRs) in tubulin expression, we analyzed mice lacking TRalpha or TRbeta compared with the wild type in both normal and TH-deprived adult animals. The results suggest that, in vivo, beta-tubulin protein expression in the liver is primarily under TRbeta positive control. In euthyroid mice lacking TRbeta, beta-tubulin expression was low. However, in the corresponding hypothyroid animals, it was found increased, therefore suggesting that the unliganded TRalpha might also upregulate beta-tubulin expression. Accordingly, TH administration to hypothyroid TRbeta-deprived mice reduced their high beta-tubulin expression. In parallel, the relatively high messenger level observed with these hypothyroid animals was reduced to the euthyroid level after T(3) treatment. The microtubular network of the mutant livers appeared, by immunofluorescence confocal microscopy, generally disorganized and drastically reduced in beta-tubulin in mice lacking TRbeta. In conclusion, our results indicate that beta-tubulin is critically controlled by TRbeta in the liver and that both TRs are probably needed to maintain the microtubular network organization of the liver.     

Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor alpha1 or beta

Thyroid hormone (T(3)) regulates bone turnover and mineralization in adults and is essential for skeletal development. Surprisingly, we identified a phenotype of skeletal thyrotoxicosis in T(3) receptor beta(PV) (TRbeta(PV)) mice in which a targeted frameshift mutation in TRbeta results in resistance to thyroid hormone. To characterize mechanisms underlying thyroid hormone action in bone, we analyzed skeletal development in TRalpha1(PV) mice in which the same PV mutation was targeted to TRalpha1. In contrast to TRbeta(PV) mice, TRalpha1(PV) mutants exhibited skeletal hypothyroidism with delayed endochondral and intramembranous ossification, severe postnatal growth retardation, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed closure of the skull sutures. Skeletal hypothyroidism in TRalpha1(PV) mutants was accompanied by impaired GH receptor and IGF-I receptor expression and signaling in the growth plate, whereas GH receptor and IGF-I receptor expression and signaling were increased in TRbeta(PV) mice. These data indicate that GH receptor and IGF-I receptor are physiological targets for T(3) action in bone in vivo. The divergent phenotypes observed in TRalpha1(PV) and TRbeta(PV) mice arise because the pituitary gland is a TRbeta-responsive tissue, whereas bone is TRalpha responsive. These studies provide a new understanding of the complex relationship between central and peripheral thyroid status.     

Differential expression of thyroid hormone receptor isoforms dictates the dominant negative activity of mutant Beta receptor

Mutations in the thyroid hormone receptor beta gene (TRbeta) cause resistance to thyroid hormone (RTH). Genetic analyses indicate that phenotypic manifestation of RTH is due to the dominant negative action of mutant TRbeta. However, the molecular mechanisms underlying the dominant negative action of mutants and how the same mutation results in marked variability of resistance in different tissues in vivo are not clear. Here we used a knock-in mouse (TRbetaPV mouse) that faithfully reproduces human RTH to address these questions. We demonstrated directly that TRbeta1 protein was approximately 3-fold higher than TRalpha1 in the liver of TRbeta(+/+) mice but was not detectable in the heart of wild-type and TRbetaPV mice. The abundance of PV in the liver of TRbeta(PV/PV) was more than TRbeta(PV/+) mice but not detectable in the heart. TRalpha1 in the liver was approximately 6-fold higher than that in the heart of wild-type and TRbetaPV mice. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed not only with TR isoforms for binding to thyroid hormone response elements (TRE) but also competed with TR for the retinoid X receptors in binding to TRE. These competitions led to the inhibition of the thyroid hormone (T(3))-positive regulated genes in the liver. In the heart, however, PV was significantly lower and thus could not effectively compete with TRalpha1 for binding to TRE, resulting in activation of the T(3)-target genes by higher levels of circulating thyroid hormones. These results indicate that in vivo, differential expression of TR isoforms in tissues dictates the dominant negative activity of mutant beta receptor, thereby resulting in variable phenotypic expression in RTH.