Severe impairment of cerebellum development in mice expressing a dominant-negative mutation inactivating thyroid hormone receptor alpha1 isoform

Thyroid hormone deficiency is known to deeply affect cerebellum post-natal development. We present here a detailed analysis of the phenotype of a recently generated mouse model, expressing a dominant-negative TRα1 mutation. Although hormonal level is not affected, the cerebellum of these mice displays profound alterations in neuronal and glial differentiation, which are reminiscent of congenital hypothyroidism, indicating a predominant function of this receptor isoform in normal cerebellum development. Some of the observed effects might result from the cell autonomous action of the mutation, while others are more likely to result from a reduction in neurotrophic factor production.     

Epigenetic regulation of phosphodiesterase 4d in restrictive cardiomyopathy mice with cTnI mutations

Epigenetic regulations play an important role in disease development. In this study, we have investigated epigenetic regulations in restrictive cardiomyopathy mice with cTnI 193 His mutation. Our results demonstrated that phosphodiesterase(PDEs) 4d was down-regulated in the heart of these mice. Further studies showed that the epigenetic modifications were associated with enhanced acetylation of histone 3 lysine 4 and lysines 9, whereas tri-methylation of histone 3 lysine 4, were decreased in histones near the PDE4d gene promoter regions. The binding levels of histone transmethylase SMYD1 and histone deacetylase HDAC1 were increased in the gene promoter regions in cTnI193 His transgenic hearts. Using immune-fluorescent labeling we found an evidence of cTnI existence in the nucleus of cardiomyocytes and Western blotting further confirmed that both wild type and mutated cTnI could be detected in the cell nucleus of the hearts. Furthermore, an interaction between cTnI and SMYD1, or cTnI and HDAC1 was observed. Overexpression of the mutated cTnI in cultured cardiomyocytes reduced the expression of PDE4d.Our data suggest that the decrease of PDE4d expression in RCM mice caused by cTnI mutations may be related to epigenetic regulation, i.e., histone acetylation and methylation, and cTnI might be involved in this procedure via an interaction with HDAC1 and SMAD1 in the hearts.     

Genomic imprinting of the type 3 thyroid hormone deiodinase gene: Regulation and developmental implications

Background

In recent years, findings in a number of animal and human models have ignited renewed interest in the type 3 deiodinase (D3), the main enzyme responsible for the inactivation of thyroid hormones. The induction of D3 in models of illness and injury has raised critical questions about the physiological significance of reduced thyroid hormone availability in those states. Phenotypes in transgenic mice lacking this enzyme also point to important developmental roles for D3. A critical determinant of D3 expression is genomic imprinting, an epigenetic phenomenon that regulates a small number of dosage-critical genes in the mammalian genome. The D3 gene (Dio3) is imprinted and preferentially expressed from one of the alleles in most tissues.

Scope of review

In the context of the physiological significance of D3 and the characteristics and purported origins of genomic imprinting, we review the current knowledge about the epigenetic mechanisms specifying gene dosage in the Dio3 locus.

Major conclusions

Altered Dio3 dosage is detrimental to development, suggesting that the level of thyroid hormone action needs to be exquisitely tailored in a timely fashion to the requirements of particular tissues. An appropriate Dio3 dosage is the result of the coordinated action of certain genomic elements and epigenetic marks in the Dlk1-Dio3 domain.

General significance

The imprinting of Dio3 prompts intriguing questions about why the level of thyroid hormone signaling should be regulated in this rare epigenetic manner, and to what extent altered Dio3 expression due to aberrant imprinting may be implicated in human conditions. This article is part of a Special Issue entitled Thyroid hormone signalling.     

A new polymorphism in the type II deiodinase gene is associated with circulating thyroid hormone parameters

Type II deiodinase (D2) is important in the regulation of local thyroid hormone bioactivity in certain tissues. D2 in skeletal muscle may also play a role in serum triiodothyronine (T(3)) production. In this study, we identified a polymorphism in the 5'-UTR of the D2 gene (D2-ORFa-Gly3Asp). We investigated the association of D2-ORFa-Gly3Asp, and of the previously identified D2-Thr92Ala polymorphism, with serum iodothyronine levels. D2-ORFa-Gly3Asp was identified by sequencing the 5'-UTR of 15 randomly selected individuals. Genotypes for D2-ORFa-Gly3Asp were determined in 156 healthy blood donors (age 46.3 +/- 12.2 yr) and 349 ambulant elderly men (age 77.7 +/- 3.5 yr) and related to serum iodothyronine and TSH levels. D2-ORFa-Asp(3) had an allele frequency of 33.9% in blood bank donors and was associated with serum thyroxine (T(4); Gly/Gly vs. Gly/Asp vs. Asp/Asp = 7.06 +/- 0.14 vs. 6.74 +/- 0.15 vs. 6.29 +/- 0.27 microg/dl, P = 0.01), free T(4) (1.22 +/- 0.02 vs. 1.16 +/- 0.02 vs. 1.06 +/- 0.04 ng/dl, P = 0.001), reverse T(3) (P = 0.01), and T(3)/T(4) ratio (P = 0.002) in a dose-dependent manner, but not with serum T(3) (P = 0.59). In elderly men, D2-ORFa-Asp(3) had a similar frequency but was not associated with serum iodothyronine levels. This new polymorphism in the 5'-UTR of D2 is associated with iodothyronine levels in blood donors but not in elderly men. We hypothesize that this might be explained by the decline in skeletal muscle size during aging, resulting in a relative decrease in the contribution of D2 to serum T(3) production.     

Dominant inhibition of thyroid hormone action selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of thyrotropin by thyroid hormone

Thyroid hormones, T4 and T3, regulate their own production by feedback inhibition of TSH and TRH synthesis in the pituitary and hypothalamus when T3 binds to thyroid hormone receptors (TRs) that interact with the promoters of the genes for the TSH subunit and TRH. All TR isoforms are believed to be involved in the regulation of this endocrine axis, as evidenced by the massive dysregulation of TSH production in mice lacking all TR isoforms. However, the relative contributions of TR isoforms in the pituitary vs. the hypothalamus remain to be completely elucidated. Thus, to determine the relative contribution of pituitary expression of TR-alpha in the regulation of the hypothalamic-pituitary-thyroid axis, we selectively impaired TR-alpha function in TR-beta null mice (TR-beta-/-) by pituitary restricted expression of a dominant negative TR-beta transgene harboring a delta337T mutation. These animals exhibited 10-fold and 32-fold increase in T4 and TSH concentrations, respectively. Moreover, the negative regulation of TSH by exogenous T3 was completely absent and a paradoxical increase in TSH concentrations and TSH-beta mRNA was observed. In contrast, prepro-TRH expression levels in T3-treated TR-beta-/- were similar to levels observed in the delta337/TR-beta-/- mice, and ligand-independent activation of TSH in hypothyroid mice was equivalently impaired. Thus, isolated TR-beta deficiency in TRH paraventricular hypothalamic nucleus neurons and impaired function of all TRs in the pituitary recapitulate the baseline hormonal disturbances that characterize mice with complete absence of all TRs.     

Role of the type 2 iodothyronine deiodinase (D2) in the control of thyroid hormone signaling

Background

Thyroid hormone signaling is critical for development, growth and metabolic control in vertebrates. Although serum concentration of thyroid hormone is remarkable stable, deiodinases modulate thyroid hormone signaling on a time- and cell-specific fashion by controlling the activation and inactivation of thyroid hormone.

Scope of the review

This review covers the recent advances in D2 biology, a member of the iodothyronine deiodinase family, thioredoxin fold‐containing selenoenzymes that modify thyroid hormone signaling in a time- and cell-specific manner.

Major conclusions

D2-catalyzed T3 production increases thyroid hormone signaling whereas blocking D2 activity or disruption of the Dio2 gene leads to a state of localized hypothyroidism. D2 expression is regulated by different developmental, metabolic or environmental cues such as the hedgehog pathway, the adrenergic- and the TGR5-activated cAMP pathway, by xenobiotic molecules such as flavonols and by stress in the endoplasmic reticulum, which specifically reduces de novo synthesis of D2 via an eIF2a-mediated mechanism. Thus, D2 plays a central role in important physiological processes such as determining T3 content in developing tissues and in the adult brain, and promoting adaptive thermogenesis in brown adipose tissue. Notably, D2 is critical in the T4-mediated negative feed-back at the pituitary and hypothalamic levels, whereby T4 inhibits TSH and TRH expression, respectively. Notably, ubiquitination is a major step in the control of D2 activity, whereby T4 binding to and/or T4 catalysis triggers D2 inactivation by ubiquitination that is mediated by the E3 ubiquitin ligases WSB-1 and/or TEB4. Ubiquitinated D2 can be either targeted to proteasomal degradation or reactivated by deubiquitination, a process that is mediated by the deubiquitinases USP20/33 and is important in adaptive thermogenesis.

General significance

Here we review the recent advances in the understanding of D2 biology focusing on the mechanisms that regulate its expression and their biological significance in metabolically relevant tissues. This article is part of a Special Issue entitled Thyroid hormone signalling.     

Absence of female-induced luteinizing hormone release in orchidectomized, sexually active mice.

This experiment was designed to determine whether the reflexive LH pulses induced in male mice by exposure to a female are dependent upon the presence of the testes. B6D2F1 males were studied because, unlike most males, these hybrids remain sexually active indefinitely after castration, demonstrating that they still recognize females and are sexually arousable. We reasoned that if LH reflexes occur independently of the testes, then exposing castrated B6D2F1 males to a female should elicit a pulse. We observed female-induced pulses in 67% of intact, naive males and 80% of intact, sexually experienced males, but they were absent in castrated, sexually experienced males. Spontaneous pulses were too frequent in castrated, naive males to distinguish potential LH reflexes. Thus, the induction of LH reflexes depends upon the presence of the testes in these hybrid males. Secondarily we found more frequent spontaneous pulses in sexually naive than in experienced mice regardless of their gonadal status. These unexpected results imply that long-term mating activity slows the pace of spontaneous LH pulses in these hybrids. Considered together, the differential effects of the testes and sexual experience on both types of LH pulses suggest that two distinct pulse-generating mechanisms exist in male mice.     

Dietary thyroid hormone replacement ameliorates hearing deficits in hypothyroid mice

Thyroid hormone (TH) insufficiency causes variable hearing impairment and mental deficiency in humans. Rodents lacking TH have congenital hearing deficiency that has been attributed to physiologic, morphologic, and developmental abnormalities of the auditory system. We examined four genetically defined strains of hypothyroid mice for development of hearing and response to TH replacement initiated during late gestation and continued through six weeks of age. Auditory brain stem response studies showed variable hearing impairment in homozygous mutants of each strain at three weeks of age relative to normal littermates. Mutants from three of the strains still had hearing deficiencies at six weeks of age. TH-enriched diet significantly improved hearing in three-week-old mutants of each strain relative to untreated mutants. Differences in the level of hearing impairment between the Prop1 ^df and Pit1 ^dw mutants, which have defects in the same developmental pathway, were determined to be due to genetic background modifier genes. Further physiologic and morphologic studies in the Cga ^tm1Sac strain indicated that poor hearing was due to cochlear defects. We conclude that TH supplement administered during the critical period of hearing development in mice can prevent deafness associated with congenital hypothyroidism of heterogeneous genetic etiology.     

A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5'-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3'-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp -100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the -2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) beta or JEG nuclear extracts (containing RXR alpha) and bacterially expressed chicken T3 receptor alpha 1 (TR alpha) can occupy both half-sites although the 3' half-site is dominant. T3 causes dissociation of TR alpha from the 5' half-site but increases binding to the 3' half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR+15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3' half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp -700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.     

Endoplasmic reticulum stress decreases intracellular thyroid hormone activation via an eIF2a-mediated decrease in type 2 deiodinase synthesis

Cells respond rapidly to endoplasmic reticulum (ER) stress by blocking protein translation, increasing protein folding capacity, and accelerating degradation of unfolded proteins via ubiquitination and ER-associated degradation pathways. The ER resident type 2 deiodinase (D2) is normally ubiquitinated and degraded in the proteasome, a pathway that is accelerated by enzyme catalysis of T(4) to T(3). To test whether D2 is normally processed through ER-associated degradation, ER stress was induced in cells that endogenously express D2 by exposure to thapsigargin or tunicamycin. In all cell models, D2 activity was rapidly lost, to as low as of 30% of control activity, without affecting D2 mRNA levels; loss of about 40% of D2 activity and protein was also seen in human embryonic kidney 293 cells transiently expressing D2. In primary human airway cells with ER stress resulting from cystic fibrosis, D2 activity was absent. The rapid ER stress-induced loss of D2 resulted in decreased intracellular D2-mediated T(3) production. ER stress-induced loss of D2 was prevented in the absence of T(4), by blocking the proteasome with MG-132 or by treatment with chemical chaperones. Notably, ER stress did not alter D2 activity half-life but rather decreased D2 synthesis as assessed by induction of D2 mRNA and by [(35)S]methionine labeling. Remarkably, ER-stress-induced loss in D2 activity is prevented in cells transiently expressing an inactive eukaryotic initiation factor 2, indicating that this pathway mediates the loss of D2 activity. In conclusion, D2 is selectively lost during ER stress due to an eukaryotic initiation factor 2-mediated decrease in D2 synthesis and sustained proteasomal degradation. This explains the lack of D2 activity in primary human airway cells with ER stress resulting from cystic fibrosis.     

Regulation of gene expression with thyroid hormone in rats with myocardial infarction

Introduction

The expression of hundreds of genes is altered in response to left ventricular (LV) remodeling following large transmural myocardial infarction (MI). Thyroid hormone (TH) improves LV remodeling and cardiac performance after MI. However, the molecular basis is unknown.

Methods

MI was produced by ligation of the left anterior descending coronary artery in female SD rats. Rats were divided into the following groups: (1) Sham MI, (2) MI, and (3) MI+T4 treatment (T4 pellet 3.3 mg, 60 days release, implanted subcutaneously immediately following MI). Four weeks after surgery, total RNA was isolated from LV non-infarcted areas for microarray analysis using the Illumina RatRef-12 Expression BeadChip Platform.

Results

Signals were detected in 13,188 genes (out of 22,523), of which the expression of 154 genes were decreased and the expression of 200 genes were increased in MI rats compared with Sham MI rats (false discovery rate (FDR) <0.05). Compared to MI rats, T4 treatment decreased expression of 27 genes and increased expression of 28 genes. In particular, 6 genes down-regulated by MI and 12 genes up-regulated by MI were reversed by T4. Most of the 55 genes altered by T4 treatment are in the category of molecular function under binding (24) and biological processes which includes immune system process (9), multi-organism process (5) and biological regulation (19) nonexclusively.

Conclusions

These results suggest that altered expression of genes for molecular function and biological process may be involved in the beneficial effects of thyroid hormone treatment following MI in rats.