3,3',5-Triiodothyroacetic acid (TRIAC) induces embryonic ζ-globin expression via thyroid hormone receptor α.

The human ζ-globin gene (HBZ) is transcribed in primitive erythroid cells only during the embryonic stages of development. Reactivation of this embryonic globin synthesis would likely alleviate symptoms both in α-thalassemia and sickle-cell disease. However, the molecular mechanisms controlling ζ-globin expression have remained largely undefined. Moreover, the pharmacologic agent capable of inducing ζ-globin production is currently unavailable. Here, we show that TRIAC, a bioactive thyroid hormone metabolite, significantly induced ζ-globin gene expression during zebrafish embryogenesis. The induction of ζ-globin expression by TRIAC was also observed in human K562 erythroleukemia cell line and primary erythroid cells. Thyroid hormone receptor α (THRA) deficiency abolished the ζ-globin-inducing effect of TRIAC. Furthermore, THRA could directly bind to the distal enhancer regulatory element to regulate ζ-globin expression. Our study provides the first evidence that TRIAC acts as a potent inducer of ζ-globin expression, which might serve as a new potential therapeutic option for patients with severe α-thalassemia or sickle-cell disease.

Analysis of Thyroid Hormone Receptor α-Knockout Tadpoles Reveals That the Activation of Cell Cycle Program Is Involved in Thyroid Hormone-Induced Larval Epithelial Cell Death and Adult Intestinal Stem Cell Development During Xenopus tropicalis Metamorphosis.

Background: There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor (TR) genes, TRα and TRß, in all vertebrates, and the expression of TRα but not TRß is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, Xenopus tropicalis undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. Methods: We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. Results: We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and TRα-knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not TRα-knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Conclusions: Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.

Thyroid Hormone Receptor Alpha Deletion in ApoE-/- Mice Alters the Arterial Renin-Angiotensin System and Vascular Smooth Muscular Cell Cholesterol Metabolism.

Thyroid hormone (TH) regulates gene transcription by binding to TH receptors (TRs). TRs regulate the genes of lipid metabolism and the renin-angiotensin system (RAS). We examined the effect of TRα deletion in ApoE-/- mice (DKO mice) on the following: (i) the expression of genes controlling cholesterol metabolism and tissue (t)RAS in the liver and aorta and (ii) the expression of these genes and the regulation of cholesterol content in cultured vascular smooth muscle cells (VSMCs). TRα deletion in ApoE-/- mice led to the repression of genes involved in the synthesis and influx of cholesterol in the liver. However, TRα deletion in the arterial wall suppressed the expression of genes involved in the esterification and excretion of cholesterol and enhanced the expression of angiotensinogen (AGT). The VSMCs of the ApoE-/- and DKO mice increased their cholesterol content during cholesterol loading, but failed to increase the expression of ATP-binding cassette transporter A1 (ABCA1). T3 addition partially corrected these abnormalities in the cells of the ApoE-/- mice but not those of the DKO mice. In conclusion, TRα deletion in ApoE-/- mice slightly increases the expression of tRAS in the aorta and aggravates the dysregulation of cholesterol content in the VSMCs.

Dual function model revised by thyroid hormone receptor alpha knockout frogs.

All vertebrates require thyroid hormone (TH) for normal growth and development. Plasma TH enters cells and alters gene expression via nuclear receptors TRα and TRß. In-vitro studies showed that TRs function as repressors of TH-inducible genes in the absence of TH and as activators of those same genes in the presence of TH. A dual function model was proposed to harmonize these molecular TR actions with the dynamic expression of TRs and peak in production of TH experienced during development. Conclusive tests of the repression activity of TRs early in development as predicted by the model awaited gene knockout technology targeting TRα. At the molecular level, active repression of genes involved in metamorphosis by TRα in the absence of TH was confirmed in whole bodies and intestine from TRα knockout studies. As a consequence of this reduced repression in TRα knockout animals, initiation of limb morphogenesis occurs precociously. However, subsequent limb development is retarded during rising plasma TH levels due to reduced TR-dependent responsivity to TH. In contrast to the limbs, intestine remodeling is delayed by one to two developmental stages in TRα knockout animals, despite de-repressed levels of TH-induced genes during premetamorphosis. Surprisingly, in the absence of TRα, hind limbs do not require gene induction by TH signaling to complete morphological growth and development, which is contrary to prediction by the dual function model. Full evaluation of the dual function model for all organs awaits the production of TRα and TRß double knockout frogs.

Hippocampal Transcriptome Profile of Persistent Memory Rescue in a Mouse Model of THRA1 Mutation-Mediated Resistance to Thyroid Hormone.

Hypothyroidism due to THRA1 (gene coding for thyroid hormone receptor α1) mutation-mediated Resistance to Thyroid Hormone (RTH) has been recently reported in human and is associated with memory deficits similar to those found in a mouse model for Thra1 mutation mediated RTH (Thra1(+/m) mice). Here, we show that a short-term treatment of Thra1(+/m) mice with GABAA receptor antagonist pentylenetetrazol (PTZ) completely and durably rescues their memory performance. In the CA1 region of the hippocampus, improvement of memory is associated with increased in long-term potentiation (LTP) and an augmentation of density of dendritic spines (DDS) onto the apical dendrites of pyramidal cells reflecting an increase in the local excitatory drive. Unbiased gene profiling analysis of hippocampi of treated Thra1(+/+) and Thra1(+/m) mice were performed two weeks and three months post treatment and identified co-expression modules that include differentially expressed genes related with and predicting higher memory, LTP and DDS in the hippocampi of PTZ-treated animals. We observed that PTZ treatment changed similar sets of genes in both Thra1(+/+) and Thra1(+/m) mice, which are known to be involved in memory consolidation and neurotransmission dynamics and could participate in the persistent effects of PTZ on memory recovery.

Thyroid hormone receptor-α deletion decreases heart function and exercise performance in apolipoprotein E-deficient mice.

The deletion of thyroid hormone receptor-α (TRα) in atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice (ApoE(-/-)TRα(0/0)) accelerates the formation of atherosclerotic plaques without aggravation of hypercholesterolemia. To evaluate other predisposition risk factors to atherosclerosis in this model, we studied blood pressure (BP) and cardiac and vascular functions, as well as exercise tolerance in young adult ApoE(-/-)TRα(0/0) mice before the development of atherosclerotic plaques. Telemetric BP recorded for 4 consecutive days showed that the spontaneous systolic BP was slightly decreased in ApoE(-/-)TRα(0/0) compared with ApoE(-/-) mice associated with a reduced locomotor activity. The percentage of animals that completed endurance (57% vs. 89%) and maximal running (0% vs. 89% at 46 cm/s speed in ApoE(-/-)TRα(0/0) and ApoE(-/-) mice, respectively) tests was lower in ApoE(-/-)TRα(0/0) mice. Moreover, during the maximal running test, both maximal running speed and running distance were significantly reduced in ApoE(-/-)TRα(0/0) mice, associated with a blunted BP response to exercise. Transthoracic echocardiography revealed a decreased interventricular septum thickness and an increased end-systolic left ventricular volume in ApoE(-/-)TRα(0/0) mice. Accordingly, left ventricular fractional shortening, ejection fraction, and stroke volume were all significantly decreased in ApoE(-/-)TRα(0/0) mice with a concomitant blunted cardiac output. No interstrain difference was observed in vascular reactivity, except that ApoE(-/-)TRα(0/0) mice exhibited an enhanced acetylcholine-induced relaxation in mesenteric and distal femoral arteries. In conclusion, the deletion of TRα in ApoE(-/-) mice alters cardiac structure and contractility; both could contribute to blunted BP response to physical exercise and impaired exercise performance.

TRα receptor mutations extend the spectrum of syndromes of reduced sensitivity to thyroid hormone.

Since 2012, eight different abnormalities have been described in the THRA gene (encoding the TRα1 thyroid hormone receptor) of 14 patients from 9 families. These mutations induce a clinical phenotype (resistance to thyroid hormone type α) associating symptoms of untreated mild congenital hypothyroidism and a near-normal range of free and total thyroid hormones and TSH (the T4/T3 ratio is nevertheless usually low). The phenotype can diversely include short stature (due to growth retardation), dysmorphic syndrome (face and limb extremities), psychoneuromotor disorders, constipation and bradycardia. The identified genetic abnormalities are located within the ligand-binding domain and result in defective T3 binding, an abnormally strong interaction with corepressors and a dominant negative activity against still functional receptors. The identification of patients with consistent phenotypes and the underlying mutations are warranted to better delineate the spectrum of the syndromes of reduced sensitivity to thyroid hormone.

Vascular function of the mesenteric artery isolated from thyroid hormone receptor-α knockout mice.

OBJECTIVE: This study evaluated the consequences of thyroid hormone receptor-α (TRα) disruption on vascular reactivity. METHODS: The activity of superior mesenteric arteries isolated from TRα knockout mice generated in the SV129 background (TRα(0/0)SV) or in a pure C57BL/6 background (TRα(0/0)C57) was compared to that of their corresponding wild-type strains (SV129 or C57BL/6 mice). RESULTS: The wild-type SV129 mice exhibited an impaired acetylcholine (Ach)-induced mesenteric artery relaxation compared to C57BL/6 mice, associated with greater responses to angiotensin II (AII) and phenylephrine (PE). The disruption of TRα decreased the vascular response to sodium nitroprusside and PE in both the SV129 and C57BL/6 genetic backgrounds. Responses to Ach and AII were also blunted, but only in TRα(0/0)C57 mice. The administration of 3,3'5-triiodo-L-thyronine sodium salt (T3) elicited a vasodilatation in C57BL/6 mice even at the lowest concentration (10(-9)M); a maximal relaxation of more than 50% was observed with the concentrations between 10(-9) and 10(-8)M. However, the response to T3 was nearly absent in TRα(0/0)C57 mice. CONCLUSION: TRα is essential for the control of vascular tone, particularly in thyroid hormone-mediated relaxation. The difference in response to Ach observed between the two wild-type mice should be taken into account for interpreting the vascular responses of genetically engineered mice.

Impaired hair growth and wound healing in mice lacking thyroid hormone receptors.

Both clinical and experimental observations show that the skin is affected by the thyroidal status. In hypothyroid patients the epidermis is thin and alopecia is common, indicating that thyroidal status might influence not only skin proliferation but also hair growth. We demonstrate here that the thyroid hormone receptors (TRs) mediate these effects of the thyroid hormones on the skin. Mice lacking TRα1 and TRß (the main thyroid hormone binding isoforms) display impaired hair cycling associated to a decrease in follicular hair cell proliferation. This was also observed in hypothyroid mice, indicating the important role of the hormone-bound receptors in hair growth. In contrast, the individual deletion of either TRα1 or TRß did not impair hair cycling, revealing an overlapping or compensatory role of the receptors in follicular cell proliferation. In support of the role of the receptors in hair growth, TRα1/TRß-deficient mice developed alopecia after serial depilation. These mice also presented a wound-healing defect, with retarded re-epithelialization and wound gaping, associated to impaired keratinocyte proliferation. These results reinforce the idea that the thyroid hormone nuclear receptors play an important role on skin homeostasis and suggest that they could be targets for the treatment of cutaneous pathologies.

TRα protects against atherosclerosis in male mice: identification of a novel anti-inflammatory property for TRα in mice.

Hypothyroidism is associated with an increased occurrence of atherosclerosis, suggesting some protective role for thyroid hormones (THs). Hypercholesterolemia is one of the major risk factor to develop this disease. Here, we show that the well-known TH cholesterol lowering effect was dependent on TH nuclear receptor (TR)ß liver activity. But most importantly, TRα was also shown to contribute of slowing down atherosclerosis progression via an independent mechanism. Introduction of TRα(0/0) deletion in the ApoE(-/-) background accelerated the appearance of plaques. Earlier cholesterol accumulation was detected in aorta macrophages, likely due to impaired cholesterol efflux. The IL-1ß inflammatory cytokine was elevated in serum and macrophages in correlation with an activation of the AKT/nuclear factor κB pathway in these cells. Inhibition of AKT prevented inflammation and restored normal cholesterol efflux. Similar low-grade inflammation was identified in TRα(0/0) male mice. Thus, the mere absence of TRα is associated with elevated levels of cytokines likely responsible for cholesterol accumulation and atherosclerosis. This TRα protective activity should be relevant for other inflammatory pathologies.

A novel role for the thyroid hormone-activating enzyme type 2 deiodinase in the inflammatory response of macrophages.

Deiodinase type 2 (D2) is a thyroid hormone-activating enzyme converting the prohormone T4 into the active hormone T3. In the present study, we show for the first time that D2 is up-regulated in the mouse liver during acute and chronic inflammation, in close correlation with the proinflammatory cytokine IL-1ß and independently of serum T3. Inflammation-induced D2 expression was confirmed in macrophages, in conjunction with selective thyroid hormone transporter (monocarboxylate transporter 10) and thyroid hormone receptor (TR)α1 stimulation, and was absent in hepatocytes. Moreover, D2 knockdown in macrophages resulted in a clear attenuation of the lipopolysaccharide (LPS)-induced IL-1ß and GM-CSF expression, in addition to aberrant phagocytosis. Locally produced T3, acting via the TRα, may be instrumental in this novel inflammatory response, because LPS-treated TRα(0/0) mice showed a markedly decreased LPS-induced GM-CSF mRNA expression. We now propose that hepatic D2 favors the innate immune response by specifically regulating cellular thyroid hormone levels in macrophages.

Impaired oxidative endoplasmic reticulum stress response caused by deficiency of thyroid hormone receptor α.

Thyroid hormone receptor α (TRα) is critical to postnatal pancreatic ß-cell maintenance. To investigate the association between TRα and the survival of pancreatic ß-cells under endoplasmic reticulum (ER) stress, the expression of endogenous TRα was inhibited by infection with an adenovirus expressing double-stranded short hairpin RNA against TRα (AdshTRα). In control adenovirus-infected pancreatic ß-cells, palmitate enhanced the expression of activating transcription factor 4 (ATF4) and heme oxygenase 1, which facilitates adaptation to oxidative ER stress. However, in AdshTRα-infected pancreatic ß-cells, palmitate did not induce ATF4-mediated integrated stress response, and oxidative stress-associated apoptotic cell death was significantly enhanced. TRα-deficient mice or wild-type mice (WT) were fed a high fat diet (HFD) for 30 weeks, and the effect of oxidative ER stress on pancreatic ß-cells was analyzed. HFD-treated TRα-deficient mice had high blood glucose levels and low plasma insulin levels. In HFD-treated TRα-deficient mice, ATF4 was not induced, and apoptosis was enhanced compared with HFD-treated WT mice. Furthermore, the expression level of 8-hydroxydeoxyguanosine, an oxidative stress marker, was enhanced in the ß-cells of HFD-treated TRα-deficient mice. These results indicate that endogenous TRα plays an important role for the expression of ATF4 and facilitates reduced apoptosis in pancreatic ß-cells under ER stress.

Type-2 iodothyronine 5'deiodinase (D2) in skeletal muscle of C57Bl/6 mice. II. Evidence for a role of D2 in the hypermetabolism of thyroid hormone receptor alpha-deficient mice.

Mice with ablation of the Thra gene have cold intolerance due to an as yet undefined defect in the activation of brown adipose tissue (BAT) uncoupling protein (UCP). They develop an alternate form of facultative thermogenesis, activated at temperatures below thermoneutrality and associated with hypermetabolism and reduced sensitivity to diet-induced obesity. A consistent finding in Thra-0/0 mice is increased type-2 iodothyronine deiodinase (D2) mRNA in skeletal muscle and other tissues. With an improved assay to measure D2 activity, we show here that this enzyme activity is increased in proportion to the mRNA and as a function of the ambient cold. The activation is mediated by the sympathetic nervous system in Thra-0/0, as it is in wild-type genotype mice, but the sympathetic nervous system effect is greater in Thra-0/0 mice. Using D2-ablated mice (Dio2-/-), we reported elsewhere and show here that, in spite of sharing a severe deficiency in BAT thermogenesis with Thra-0/0 and UCP1-knockout mice, they do not have an increase in oxygen consumption, and they gain more weight than wild-type controls when fed a high-fat diet. UCP3 mRNA is highly responsive to thyroid hormone, and it is increased in Thra-0/0 mice, particularly when fed high-fat diets. We show here that muscle UCP3 mRNA in hypothyroid Thra-0/0 mice is responsive to small dose-short regimens of T(4), indicating a role for locally, D2-generated T(3). Lastly, we show that bile acids stimulate not only BAT but also muscle D2 activity, and this is associated with stimulation of muscle UCP3 mRNA expression provided T(4) is present. These observations strongly support the concept that enhanced D2 activity in Thra-0/0 plays a critical role in their alternate form of facultative thermogenesis, stimulating increased fat oxidation by increasing local T(3) generation in skeletal muscle.

Thyroid hormone receptor ß mediates thyroid hormone effects on bone remodeling and bone mass.

Excess thyroid hormone (TH) in adults causes osteoporosis and increases fracture risk. However, the mechanisms by which TH affects bone turnover are not elucidated. In particular, the roles of thyroid hormone receptor (TR) isotypes in the mediation of TH effects on osteoblast-mediated bone formation and osteoclast-mediated bone resorption are not established. In this study we have induced experimental hypothyroidism or hyperthyroidism in adult wild-type, TRα- or TRß-deficient mice and analyzed the effects of TH status on the structure and remodeling parameters of trabecular bone. In wild-type mice, excess TH decreased bone volume and mineralization. High TH concentrations were associated with a high bone-resorption activity, assessed by increased osteoclast surfaces and elevated concentrations of serum bone-resorption markers. Serum markers of bone formation also were higher in TH-treated mice. TRα deficiency did not prevent TH action on bone volume, bone mineralization, bone formation, or bone resorption. In contrast, TRß deficiency blocked all the early effects of excess TH observed in wild-type mice. However, prolonged exposure to low or high TH concentrations of TRß-deficient mice induced mild modifications of bone structure and remodeling parameters. Together our data suggest that TRß receptors mediate the acute effects produced by transient changes of TH concentrations on bone remodeling, whereas TRα receptors mediate long-term effects of chronic alterations of TH metabolism. These data shed new light on the respective roles of TRs in the control of bone metabolism by TH.

Thyroid hormone receptor α and regulation of type 3 deiodinase.

Mice deficient in thyroid hormone receptor α (TRα) display hypersensitivity to thyroid hormone (TH), with normal serum TSH but diminished serum T(4). Our aim was to determine whether altered TH metabolism played a role in this hypersensitivity. TRα knockout (KO) mice have lower levels of rT(3), and lower rT(3)/T(4) ratios compared with wild-type (WT) mice. These alterations could be due to increased type 1 deiodinase (D1) or decreased type 3 deiodinase (D3). No differences in D1 mRNA expression and enzymatic activity were found between WT and TRαKO mice. We observed that T(3) treatment increased D3 mRNA in mouse embryonic fibroblasts obtained from WT or TRßKO mice, but not in those from TRαKO mice. T(3) stimulated the promoter activity of 1.5 kb 5'-flanking region of the human (h) DIO3 promoter in GH3 cells after cotransfection with hTRα but not with hTRß. Moreover, treatment of GH3 cells with T(3) increased D3 mRNA after overexpression of TRα. The region necessary for the T(3)-TRα stimulation of the hD3 promoter (region -1200 to -1369) was identified by transfection studies in Neuro2A cells that stably overexpress either TRα or TRß. These results indicate that TRα mediates the up-regulation of D3 by TH in vitro. TRαKO mice display impairment in the regulation of D3 by TH in both brain and pituitary and have reduced clearance rate of TH as a consequence of D3 deregulation. We conclude that the absence of TRα results in decreased clearance of TH by D3 and contributes to the TH hypersensitivity.

Erythroid defects in TRalpha-/- mice.

Thyroid hormone and its cognate receptor (TR) have been implicated in the production of red blood cells. Here, we show mice deficient for TRalpha have compromised fetal and adult erythropoiesis. Erythroid progenitor numbers were significantly reduced in TRalpha(-/-) fetal livers, and transit through the final stages of maturation was impeded. In addition, immortalized TRalpha(-/-) erythroblasts displayed increased apoptosis and reduced capacity for proliferation and differentiation. Adult TRalpha(-/-) mice had lower hematocrit levels, elevated glucocorticoid levels, and an altered stress erythropoiesis response to hemolytic anemia. Most TRalpha(-/-) animals contained markedly altered progenitor numbers in their spleens. Strikingly, 20% of TRalpha(-/-) mice failed to elicit a stress erythropoiesis response and recovered very poorly from hemolytic anemia. We conclude that an underlying erythroid defect exists in TRalpha(-/-) mice, demon-strating the importance of TRalpha to the erythroid compartment.

Thyroid status during skeletal development determines adult bone structure and mineralization.

Childhood hypothyroidism delays ossification and bone mineralization, whereas adult thyrotoxicosis causes osteoporosis. To determine how effects of thyroid hormone (T3) during development manifest in adult bone, we characterized TRalpha1(+/m)beta(+/-) mice, which express a mutant T3 receptor (TR) alpha1 with dominant-negative properties due to reduced ligand-binding affinity. Remarkably, adult TRalpha1(+/m)beta(+/-) mice had osteosclerosis with increased bone mineralization even though juveniles had delayed ossification. This phenotype was partially normalized by transient T3 treatment of juveniles and fully reversed in compound TRalpha1(+/m)beta(-/-) mutant mice due to 10-fold elevated hormone levels that allow the mutant TRalpha1 to bind T3. By contrast, deletion of TRbeta in TRalpha1(+/+)beta(-/ -) mice, which causes a 3-fold increase of hormone levels, led to osteoporosis in adults but advanced ossification in juveniles. T3-target gene analysis revealed skeletal hypothyroidism in TRalpha1(m/+)beta(+/-) mice, thyrotoxicosis in TRalpha1(+/+)beta(-/-) mice, and euthyroidism in TRalpha1(+/)beta(-/-) double mutants. Thus, TRalpha1 regulates both skeletal development and adult bone maintenance, with euthyroid status during development being essential to establish normal adult bone structure and mineralization.

Thyroid hormone excess rather than thyrotropin deficiency induces osteoporosis in hyperthyroidism.

Thyrotoxicosis is an important but under recognized cause of osteoporosis. Recently, TSH deficiency, rather than thyroid hormone excess, has been suggested as the underlying cause. To investigate the molecular mechanism of osteoporosis in thyroid disease, we characterized the skeleton in mice lacking either thyroid hormone receptor alpha or beta (TRalpha(0/0), TRbeta-/-). Remarkably, in the presence of normal circulating thyroid hormone and TSH concentrations, adult TRalpha(0/0) mice had osteosclerosis accompanied by reduced osteoclastic bone resorption, whereas juveniles had delayed endochondral ossification with reduced bone mineral deposition. By contrast, adult TRbeta-/- mice with elevated TSH and thyroid hormone levels were osteoporotic with evidence of increased bone resorption, whereas juveniles had advanced ossification with increased bone mineral deposition. Analysis of T3 target gene expression revealed skeletal hypothyroidism in TRalpha(0/0) mice, but skeletal thyrotoxicosis in TRbeta-/- mice. These studies demonstrate that bone loss in thyrotoxicosis is independent of circulating TSH levels and mediated predominantly by TRalpha, thus identifying TRalpha as a novel drug target in the prevention and treatment of osteoporosis.

Behavioral inhibition and impaired spatial learning and memory in hypothyroid mice lacking thyroid hormone receptor alpha.

Thyroid hormone insufficiency leads to impaired neurogenesis, behavioral alterations and cognitive deficits. Thyroid hormone receptors, expressed in brain regions involved in these behaviors, mediate the effects of thyroid hormone deficiency or excess. To determine the contribution of thyroid hormone receptor alpha (TRalpha) in these behaviors, we examined the behavior of euthyroid as well as hypo- and hyperthyroid mice lacking all isoforms of the TRalpha (TRalpha(o/o)). The hypothyroxinemic TRalpha(o/o) mice demonstrated behavioral inhibition, manifested in decreased activity and increased anxiety/fear in the open field test (OFT) and increased immobility in the forced swim test (FST) compared to C57BL/6J mice. TRalpha(o/o) mice also showed learning and recall impairments in the Morris water maze (MWM), which were exaggerated by hypothyroidism in TRalpha(o/o) mice. These impairments were concurrent with increased thigmotaxis, suggesting an increased anxiety-like state of the TRalpha(o/o) mice in the MWM. Expression of genes, known to be involved in processes modulating learning and memory, such as glucocorticoid receptor (GR), growth-associated protein 43 (GAP-43) and neurogranin (RC3), were significantly decreased in the hippocampus of TRalpha(o/o) mice. GR expression was also decreased in the frontal cortex and amygdala of TRalpha(o/o) mice, indicating that expression of GR is regulated, probably developmentally, by one or more isoforms of TRalpha in the mouse brain. Taken together these data demonstrate behavioral alterations in the TRalpha(o/o) mice, indicating the functional role of TRalpha, and a delicate interaction between TRalpha and TRbeta-regulated genes in these behaviors. Thyroid hormone-regulated genes potentially responsible for the learning deficit found in TRalpha(o/o) mice include GR, RC3 and GAP-43.

Regulation of nuclear coactivator and corepressor expression in mouse cerebellum by thyroid hormone.

Thyroid hormone (TH) has an important role in central nervous system development. TH action is mediated by a number of transcription factors including thyroid hormone receptors (TRs) in combination with a group of coregulators that can either activate (coactivators) or repress (corepressors) transcription in the presence of TH. The aims of this report were to determine if regulation of the corepressor Hairless (Hr) by TH was TR-isoform- mediated in neonatal cerebellum and to determine if other cerebellar corepressors (SMRT and NCoR) and coactivators (SRC family) are also regulated by TH. In order to study this we examined 14-day-old and adult knockout mice that lack expression of the TRbeta or TRalpha isoforms and measured mRNA expression in untreated, hypothyroid and TH-treated young mouse pups. TH-treated wild-type and TRbeta-deficient mice demonstrated upregulation of Hr by 22.8- +/- 8.6- and 11.8- +/- 3.6-fold respectively, which was not upregulated in TRalpha-deficient mice. In wild-type mice, TH treatment results in a reciprocal decrease (61%) in the coactivator SRC-1. These changes were not observed in adult mouse cerebellum. No effect was seen with NCoR and SRC-3 expression. SMRT was 3-fold increased in TH treatment of only wild-type mouse pups. We conclude that (1) TRalpha is the major TR regulating Hr expression in the cerebellum of young mouse pups; (2) TH upregulates Hr and SMRT and downregulates SRC-1; (3) NcoR and SRC-3 may not be regulated by TH in the cerebellum at the transcriptional level; and (4) autoregulation of TH action may be mediated through TH-dependent expression of the cofactors necessary for TH action in the cerebellum and may be developmentally specific.