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Thyroid Hormones Signaling Is Getting More Complex: STORMs Are Coming

Laboratory of Molecular Cell Biology (F.F., K.G., J.S.), Unité Mixte de Recherche, Centre National de la Recherche Scientifique 5161, Institut National de la Recherche Agronomique 1237, Ecole Normale Supérieure de Lyon, Institut Fédératif de Recherche 128 Biosciences Lyon Gerland, and Université Claude Bernard Lyon 1 (J.S.), 69364 Lyon Cedex 07, France

Address all correspondence and requests for reprints to: Frederic Flamant, Laboratory of Molecular Cell Biology, Unité Mixte de Recherche, Centre National de la Recherche Scientifique 5161, Institut National de la Recherche Agronomique 1237, Ecole Normale Supérieure de Lyon, Institut Fédératif de Recherche 128 Biosciences Lyon Gerland, 46 allée d’Italie, 69364 Lyon Cedex 07, France. E-mail: Frederic.Flamant{at}ens-lyon.fr.

	ABSTRACT

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
T₃ regulates many physiological and developmental processes by binding to thyroid hormone receptors (TRs). This induces a conformational change of DNA-bound TRs that releases corepressors in favor of coactivators. The associated chromatin modifications induce polymerase II recruitment. Mouse genetic studies clarified the respective contribution of each receptor isoform and revealed the important activity of unliganded TRs. They also confirm the paradoxical negative regulation of some promoters by liganded TRs. Recent advances place these molecular events in a broader context of extra- and intracellular regulation: control of ligand availability, changes in the cell sensitivity to T₃, nongenomic effects, and cross talks with other signaling pathways contribute to increase the diversity and complexity of thyroid hormones signaling. A promising novel class of TRs synthetic ligands, called STORMs (selective TR modulators), might allow for tissue- and promoter-specific interventions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
T₃, THE ACTIVE FORM of thyroid hormone (TH) and its biologically less potent precursor, T₄, are both secreted by the thyroid gland. Most T₃ synthesis results from T₄ deiodination in the periphery. THs exert pleiotropic effects on fetal and postnatal development as well as homeostatic regulation in adults (1). TRH, secreted by the hypothalamus, controls pituitary TSH production, which in turn activates TH production and secretion from the thyroid gland. Negative feedback regulation exerted by TH on TRH and TSH production ensures remarkable stability of serum TH levels. Early binding studies revealed the existence of nuclear receptors for TH (2) and suggested that T₃, and to a lesser extent T₄, act by binding to these receptors. In line with these observations, the biological activities of other natural iodinated metabolites correlated with their receptor binding affinities. Triiodothyroacetic acid has both high binding affinity and activity, whereas rT₃ binds TR with low affinity and has low potency.

In 1986, the search for a cellular protooncogene related to the avian v-erbA oncogene revealed the existence of two genes, now called THRA and THRB, which encode the nuclear receptors for T₃. This discovery confirmed that T₃ acts directly on transcription and paved the way for detailed study of the molecular mechanisms for T₃ signaling. In mammals, there are three different thyroid hormone receptors (TRs), TR1, which is encoded by the THRA gene, and TRß1 and TRß2, encoded by the THRB gene [a fourth TRß3 isoform seems to be rat specific (3)]. These receptors have a very similar structure, containing a central DNA-binding domain and a C-terminal domain, which activates transcription upon ligand binding.

TR agonists could potentially serve as pharmacological agents with beneficial effects, such as lowering body weight and serum cholesterol level. However, an excess of TH causes side effects, such as bone and muscle loss, tachycardia, and atrial arrhythmia. The possibility for synthetic TR ligands to exert beneficial effect without adverse reactions makes TR very attractive targets for new drug development. By analogy with selective estrogen receptor modulators (4), there is hope that new TR ligands will allow for tissue- and promoter-specific interventions. We propose to call this novel class of drugs selective thyroid hormone receptor modulators (STORMs).

THs transmit diverse signals that can vary in a tissue- and gene-dependent manner, and TR can activate or repress target gene expression. This review is an attempt to place our basic knowledge of TR-mediated transcriptional control into the broader context of intracellular and extracellular regulation.

	TRs ARE LIGAND-REGULATED TRANSCRIPTION FACTORS (FIG. 1)

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
The general model for transcriptional regulation by nuclear receptors (3) applies to TRs with several notable features. TRs bind DNA at T₃ response elements (TREs) in the promoter of target genes mainly as heterodimers with retinoic X receptors (RXRs). TR/RXR heterodimers activate transcription mainly on DR4 elements (5'-AGGTCANNNNAGGTCA-3'), everted repeats spaced by three or six nucleotides (5'-TGACCTNNNAGGTCA-3' 5'-TGACCTNNNNNNAGGTCA-3') and on palindromic elements (5'-AGGTCATGACCT-3'). Up to now, this last element has been identified only in the context of the GH gene promoter, which possesses a third half-site, creating a DR4. The relevance of the palindromic TRE to T₃ regulation is thus unclear. These canonical half-site sequences and arrangements are not the only functional TREs, as many natural TREs do not fit in these categories (5). In contrast to steroid hormone receptors, TR DNA binding is not ligand dependent. Although it has been thought that RXR played a silent role in activation, recent studies indicate that, at least in some cases, the RXR ligand, 9-cis-retinoic acid, can activate TR/RXR heterodimers (6, 7, 8). In the absence of ligand the receptors directly recruit ATP-dependent chromatin-remodeling complexes [chromatin-remodeling complexes (CRCs) associating SWI/SNF/NURD/C-terminal binding protein], exchange proteins [transducin-like 1 (TBL1)/TBL1 receptor (TBLR1)] (9), and basal corepressors such as nuclear receptor corepressor and silencing mediator of retinoid and thyroid hormone receptor, which function as platforms for the further recruitment of several other complexes with histone deacetylase activity. In this situation the activity of the targeted promoter is much lower than in the absence of nuclear receptor binding, a phenomenon referred to as "basal transcriptional repression" or "silencing" (10).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Regulation of Gene Expression by TRs: A Simplified View Regulation of a target gene with a positive TRE in three different situations. TR is bound to TRE either as a homodimer or as heterodimer with RXR. In the absence of ligand, it recruits corepressor molecules (CoR) that, in turn, recruits other complexes (HMT, histone methyl transferase; HDAC, histone deacetylase; HDM, histone demethylase) and interfere with the basal transcription machinery, leading to repression. In the knockout situation, other regulatory pathways ensure a basal transcription level. T3 binding to TR induces the release of corepressors, recruitment of coactivators (CoA) that, in turn, recruit other complexes (HMT, histone arginine methyltransferase; HAT, histone acetyl-transferase; CRC, chromatin remodeling complex), which directly interact with the basal transcription machinery and lead to increased promoter activity (thick dark arrow).

Although widely accepted, and sometimes regarded as dogma, this model has several insufficiencies. First, the hypothesis is likely to be an oversimplification, because it does not take into account more than half of the documented T₃-dependent and T₃-independent protein-protein interactions. For example, TRs interact with cyclinD1 (15), the TFIIH p62 subunit (16), a noncoding RNA called "SRA" (steroid receptor RNA activator) (17), and the peroxisome proliferator-activated receptor- (PPAR) coactivator 1 (18). Second, it does not explain how the action of TR can be specific, because DR4 elements are also recognized by a number of other nuclear receptors including RXR, retinoid-related orphan receptor, liver X receptor (LXR), pregnane X receptor, constitutive androstane receptor (CAR), and TR4. Although Hairless (19) and Alien (20) are two corepressors that interact with only a handful of nuclear receptors, TR specificity cannot be explained by the existence of unique coregulator complexes. Third, the model provides no explanation for T₃-mediated gene repression, or activation by unliganded TR, which are probably of major importance for the negative feedback regulation of TRH and TSH in vivo. Finally, it does not take into account some controversial in vitro observations of the formation of TR trimers (21), RAR/TR (22) and chicken ovalbumin upstream promoter transcription factor/TR heterodimers (23).

	OTHER LEVELS OF REGULATION: LIGAND AVAILABILITY

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
Thyrocytes mainly secrete T₄, the low-affinity precursor of T₃. Deiodinases are selenoproteins that catalyze the conversion of T₄ into T₃ (type 1 and type 2 deiodinases) and T₃ catabolism (type 1 and type 3 deiodinases). The three corresponding genes display a complex expression pattern with opposing regulation by TH: hypothyroidism increases type 2 deiodinase gene expression in brain, whereas hyperthyroidism increases type 1 deiodinase in liver and type 3 deiodinase in brain. Knockout of these genes fails to completely deregulate TH levels (24, 25) whereas a mutation in the human SECISBP2 gene, which encodes a common regulator of selenoprotein translation, results in low circulating level of T₃ (26). This suggests that TH levels are regulated by other unidentified selenoproteins and reinforces the view that complex TH metabolism, in addition to the role of deiodinases, should not be overlooked (27).

Transmembrane passage of THs has been thought to be passive, given their lipophilic nature. However, it is now evident that TH uptake is transporter and energy dependent (28) and that intracellular TH levels are highly variable in tissues (29). The identification of TH transporters has been a challenging task. Several transporters with broad specificity, such as Ntcp/Slc10a1, organic anion transporting polypeptides (OATPs), and amino acid transporters [large neutral amino acids transporter (LAT1 and LAT2)], are able to transport TH, but their weak affinity makes their relevance to TH transport inconclusive. By contrast, one of the OATP family members, OATPC1 (also called OATP-F, OATP14, SLC21A14, or SLC01C1) displays high affinity for both T₄ and rT₃ (normally a poor ligand for TR), but not for T₃. OATPC1 allows for both import and efflux of these molecules (30). The expression of the OATPC1 gene is positively regulated by T₃ and is abundant in capillaries throughout the brain. These features suggest that OATPC1 may be an important transporter of T₄ across the blood-brain barrier. Imported T₄ is then locally converted into T₃ by type 2 deiodinase, protecting the brain against low serum T₃ level. MCT8 is another transporter with high affinity for T₄, T₃, rT₃, and T₂ in rodents (31); the human version is more specific for T₃. The MCT8 gene is expressed in a number of tissues including skeletal muscle, neurons, and most strongly in liver. Patients with MCT8 mutations show increased level of circulating T₃, with mildly decreased T₄, normal TSH levels, and severe psychomotor retardation (32, 33, 34). This phenotype suggests that the lack of T₃ transport from astrocytes to neurons causes this defect, whereas other alternate transport pathways can compensate outside of the brain.

	OTHER LEVELS OF REGULATION: ALTERED CELL SENSITIVITY TO TH

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
In principle, there are several ways to change cellular sensitivity to TH, i.e. to modify the amplitude of the response to a given T₃ concentration. In particular, TR target gene regulation may integrate extracellular signals and may not only reflect variations in the intracellular concentration of TH. One obvious possible mechanism would be the regulation of the level of expression of the THRA and THRB genes by extracellular signals, which would indirectly determine the promoter occupancy of TR-target genes. A good example of this phenomenon is TH-induced Xenopus laevis metamorphosis, in which increased promoter occupancy by TR depends on the induction of THRB expression (35). In mammals, TR1 mRNA level drops by 80% in only 4 h when macrophages are activated by lipopolysaccharides, and -interferon quickly up-regulates both THRA and THRB (36), suggesting that acute changes in T₃ sensitivity occur in this cell type. T₃ itself can change the half-life of TR1 but not TRß1 mRNA in cardiomyocytes (37). It is currently not known whether this observation can be extended to other contexts. Another possible level of regulation is TR mRNA translation, which can also be regulated during the cell cycle (38). Finally, TR protein levels in the nucleus can be modified through alterations in nuclear transport (reviewed in Ref. 39). The nuclear localization of a green fluorescent protein-TRß fusion protein is constitutive. However, amino acid changes, which prevent interaction with nuclear receptor corepressor, render nuclear localization ligand dependent. Furthermore, an excess of RXR can shift the distribution of this mutant receptor from cytoplasmic to nuclear. This suggests that the nuclear localization of unliganded TRß results from interactions with corepressors and RXR (40, 41). TRß1 has been shown to be acetylated and phosphorylated in its DNA-binding domain (42, 43). This last modification correlates with nuclear localization (44). Finally, TR-containing regulatory complexes can be destabilized by the p23 chaperone (45).

Recent data show that the TR-DNA interaction appears to be highly dynamic, as is the case for estrogen receptors (46). Liganded TRs seem to occupy TREs only transiently and recruit coactivators in a cyclical manner over the 2 h examined (47). Interestingly, each of the four target genes, which have been studied so far only in pituitary GH₃ cells, has a particular temporal recruitment pattern of coactivators. Previous TR chromatin immunoprecipitation studies did not resolve promoter occupancy at the same time scale and failed to reveal cyclical recruitment by TR (9, 48). Whether the cyclical recruitment of coactivators by TR can be generalized to other cell types and other TR target genes remains to be determined. It has also been reported that TR, like other nuclear receptors (49), is rapidly degraded by the proteasome in GH₃ pituitary cells (50) and cardiomyocytes (37) after T₃ binding. TR transcriptional activity can also be antagonized by THRA isoforms, in a poorly understood manner. Alternate splicing and promoter usage enable THRA to encode three isoforms [TR2 (51), TR1, and TR2 (52), which act as antagonists of T₃-mediated gene activation; the presence of the TR6 isoform, recently described in mice (53), remains to be verified in other mammalian species]. The tissue expression level of these isoforms is variable among tissues and could influence the cell sensitivity to T₃. Of note, TR1 and TR2 are unable to bind DNA but can trigger TR1 degradation (54).

	OTHER LEVELS OF REGULATION: DO THs ACT WITHOUT BINDING TO TR?

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
Whereas there is no doubt that THs act directly on transcription via nuclear TRs, it is possible that other pathways exist. TH could act on TR-independent pathways or modulate TR transcriptional function without binding to TREs. Although these mechanisms have been controversial, recent studies have identified some potential alternate pathways (55). A number of experimental systems have revealed unconventional actions of TH, called, perhaps improperly, "nongenomic effects." For example, TH can act at physiological concentrations on kinase activities, synaptic Ca⁺⁺ influx, and actin polymerization in cell-free, nuclei-free systems (56, 57, 58). The rapid response observed on synaptic components has led to the proposal that THs function as neurotransmitters (59). THs are also able to promote ion efflux from Ca⁺⁺ overloaded cultured cardiomyocytes within 10 sec (60). This effect has been proposed to be driven by T₃ binding to the mitochondrial p43 isoform. The translation of this fifth TR isoform is initiated from an alternate downstream start codon on the TR1 mRNA. It does not interfere with TR nuclear function but seems to be targeted to mitochondria where it might act on mitochondrial gene transcription (61, 62). The mechanism would thus be nonnuclear but would still require the expression of THRA.

Adding another level of complexity, TR-independent and TR-dependent TH responses can intersect because cytoplasmic actions of TH could change cellular sensitivity to TH and thereby enhance TR nuclear activity. It has been proposed recently that TR acetylation, a process that seems to be important for coactivator recruitment and appears to depend on the MAPK pathway, can be rapidly stimulated by both T₃ and T₄ (42) after binding at the cell surface to the Vß3 integrin (63). A related, but different, conclusion was reached after a recent transcriptome study of human fibroblasts identified hypoxia-inducible factor (HIF)-1 as a major T₃ direct target gene. This transcriptional activation was blocked by a phosphatidylinositol-OH-3-kinase (PI3K) inhibitor but not by a MAPK inhibitor (64, 65). The mechanism of T₃ modulation of this pathway has been interpreted as resulting from a direct interaction between the PI3K-regulatory subunit p85 and TRß1 (66). Interestingly, the regulation of intracellular Ca⁺⁺ wave periodicity in Xenopus oocytes is another PI3K-dependent effect of TH that was found to rely upon the expression of exogenous TRß1, but did not depend on its DNA binding activity (67).

Although genetic evidence for the physiological relevance of these effects is lacking, it is clear that T₃ metabolites, which cannot bind TR, possess intrinsic biological activity. T₂, which results from T₃ deiodination and is usually considered to be a degradation product, seems to act in mitochondria (68). 3,5-Diiodothyropropionic acid is more potent than T₃ and T₄ in promoting MAPK-dependent pathway activation and angiogenesis via binding to Vß3 integrin (69). More convincingly, decarboxylation of T₃ gives rise to thyronamines (T1AM and T0AM), which bind to the membrane G protein-coupled receptor TAR1 but not to TR and display a rapid and strong effect on heart rate and body temperature (70).

	CROSS TALK: TR FUNCTIONS WITHOUT BINDING TO DNA?

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
TRs have been implicated in cross-regulation with other nuclear receptors. First, RXR is not always a silent partner in heterodimers (6, 8). Second, TR/RAR heterodimers might be rather stable under some circumstances and, depending on the response elements, may respond to retinoic acid (22). Interference with other nuclear receptors such as LXR (71, 72) and PPAR (73) is likely due to competition between RXR/TR, RXR/LXR, and RXR/PPAR heterodimers for binding to hormone response elements. TR facilitates liganded estrogen receptor transcriptional activity in a more complex way, involving estrogen receptor phosphorylation (74, 75).

Other cross talk mechanisms are independent of TR binding to DNA. Liganded TRs antagonize activator protein 1 (AP1) activation of the type 3a collagenase gene (76). The activity of c-Jun, a component of the AP1 complex, is potentiated by amino-terminal phosphorylation mediated by the Jun amino-terminal kinase, modifications that are required to recruit the transcriptional coactivator CREB-binding protein. Liganded TRs block this pathway by inhibiting Jun amino-terminal kinase activity (77). Unlike glucocorticoid receptor/AP1 cross talk, TR/AP1 cross talk is not released by glucocorticoid receptor-interacting protein 1 coactivator overexpression and does not involve a reduction in AP1 DNA binding capacity (78). Direct interaction of liganded TR with CREB provides an explanation for the negative effect of T₃ on CREB-mediated gene expression and, conversely, the negative effect of CREB on T₃-mediated transcription (79, 80, 81). Finally, when TR binding sites are located adjacent to CCCTC-binding factor (CTCF)-binding sites, TR can abrogate the insulating function of CTCF. This finding has led to the radical proposal that TR can regulate gene expression over long distances without acting directly on transcription initiation (82).

	RESPECTIVE FUNCTIONS OF TH-BINDING TR ISOFORMS

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
A large set of mutations have been targeted to the THRA or THRB loci in mice by homologous recombination, resulting in the generation of many strains harboring various combinations of deleted isoforms (Table 1). Although the functions of the isoforms that do not bind T₃ (TR1, TR2, and TR2) remain obscure (83, 84, 85), comparative analysis greatly clarifies the status of the three T₃ binding isoforms: TR1, TRß1, and TRß2. These receptors behave similarly in most in vitro assays, although TRß2 possesses a supplementary ligand-independent activation domain at its N terminus (86), and, in the absence of ligand, forms very stable homodimers on a DR4 response element (87). By contrast, the consequences of THRA and THRB mutations are very different. Whether these differences reflect dissimilarities in the receptors’ intrinsic properties, or of their expression patterns is still being debated.

Compound knockout mice, which are devoid of all receptors, were generated by crossing THRA and THRB knockout mice (54, 105). They were viable and some mice were fertile. They displayed accentuated phenotypic alterations and dysregulation of the hypothalamic-pituitary-thyroid axis as compared with single knockout mice (94, 106, 107). For example, TSH and TH levels reached extreme levels, indicating a complete loss of feedback regulation These findings support the view that, in some tissues, the three TR isoforms might have similar functions and that the contribution by the respective receptors in a given cell type is dictated mainly by their respective abundance. Microarray analysis of liver RNA of TR and/or TRß knockout mice also suggests that TR isoform-specific regulation of target genes is rare (108). Studies on the related retinoic acid receptors, combining isoform-specific ligands and knockouts, revealed, however, that apparent functional redundancy between isoforms might, in part, be an artifact of the knockout models (109).

	DO UNLIGANDED RECEPTORS REGULATE GENE EXPRESSION IN PATHOLOGICAL AND PHYSIOLOGICAL CONTEXTS?

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
In transfected cells, unliganded TRs exert strong basal transcriptional repression (110, 111). The influence of unliganded TR in pathological or physiological situations is under current investigation and remains controversial. A first clue for an important role of unliganded TR in the manifestations of hypothyroidism comes from the observation that mice devoid of all TR isoforms (54, 105) have a less severe phenotype than Pax8^–/– mice. The latter mice have thyroid agenesis, resulting in deep congenital hypothyroidism, which is lethal within 3 wk after birth (112). Microarray analysis confirms the difference between hypothyroid and THRA/THRB knockout mice and identifies genes for which basal expression is repressed in hypothyroid livers (108). These results suggest that the unliganded TRs present in Pax8^–/– mice but not in THRA/THRB knockout mice, are detrimental to postnatal development and repress liver gene expression. The developmental consequences of TH depletion are attenuated when TR1 is absent (113, 114), supporting the conclusion that the unliganded TR1 isoform is deleterious. In line with this hypothesis, the consequences of THRA deletion are limited compared with those observed in knock-in mutations. Three knock-in mutations of TR1 have been introduced into the ligand-binding domain, with distinct effects on ligand binding and cofactor interactions. However, all these mutations preserve DNA binding and reduce TR1 transactivation. Mice heterozygous for these dominant mutations display many features of congenital hypothyroidism, suggesting a constitutive repression of T₃ target genes (115, 116, 117, 118).

Unliganded TR1 also seems to repress gene expression during normal fetal heart development. At early stages, when the fetal T₃ level is very low, TR1 represses the cardiac expression of genes that it will activate after birth, in response to increased circulating T₃ levels. The transcriptional switch that occurs between these two developmental stages parallels the increase of heart rate (119). Direct T₃ measurement (120), observation of reporter gene expression pattern (29), and local changes of retinoid-related orphan receptor- expression pattern in mice lacking all TR expression (121) suggest that unliganded receptors are present in some brain areas after birth.

Although these data provide good evidence for a repressive action of TR1, in both pathological and physiological situations, they remain inconclusive for several reasons. First, mice without TR have very high levels of TH due to a loss of feedback regulation. If TH were to act on development in a TR-independent manner, as recently proposed (122), the comparison between Pax8^–/– hypothyroid mice and mice without TR would be interpreted differently. Second, unlike the Pax8^–/–TR^0/0 mice, the Pax8^–/–TR1^–/– mice in which TR2 expression is maintained have a lethal phenotype (123). A more precise phenotypic comparison between Pax8^–/–, Pax8^–/–TR^0/0, Pax8^–/–TR1^–/– and THRA knock-in mice is required to decide whether unliganded TR1 is the only isoform responsible for the various phenotypes.

At the present time, there is no convincing in vivo evidence for a developmental role of unliganded TRß. For example the phenotype of Pax8^–/–TRß^–/–mice is very similar to the one of Pax8^–/– mice (113), and a TRß-specific ligand failed to rescue cerebellar development in hypothyroid mice (114). Microarray analysis supports the conclusion that there are very few genes (i.e. Bcl3, Tapbp, and lysozyme) that can be down-regulated by unliganded TRß1 in liver (108). Several knock-in mice have been generated with mutations in the C-terminal activation domain of TRß, which lock the TRß1 and TRß2 receptors in a constitutively repressive conformation. This results in a phenotype similar to the human genetic syndrome of resistance to thyroid hormone (RTH). Unlike what has been observed with TR, these mutations do not appear to affect development and homeostasis more than the THRB knockouts. One noticeable exception is cerebellar development, which is severely disturbed in one of these mouse lines (124).

	UPSIDE-DOWN-REGULATION: TSH DOWN-REGULATION BY LIGANDED TRß1 AND TRß2 REQUIRES AN INTACT DNA-BINDING DOMAIN AND COACTIVATOR RECRUITMENT

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
Negative transcriptional regulation by liganded TR of the TSH and TSHß genes in the pituitary, and of the TRH gene in the hypothalamus, is critical for TH feedback regulation. However the mechanism underlying this regulation cannot be explained by the current model of TR function. Negative regulation by TR might be much more prevalent than previously thought, because transcriptome analyses reveal that liganded TR exert, directly or indirectly, both positive and negative regulation within the same cell of many target genes (108). Conversely, there are also indications that unliganded TR can activate transcription on specific response elements and through a molecular mechanism that is likely to be different (125, 126, 127).

There is no entirely satisfying cellular model to study TRH, TSH, and TSHß regulation, although the endogenous TSHß gene is down-regulated by T₃ in the pituitary cell line TT1 (128). The use of several artificial models has brought confusion and controversy. For example the role of the so-called "negative TREs" in promoters is not clearly established, and the requirement for DNA binding by TR is under dispute. In this context, recent mouse genetics analyses have attempted to resolve the controversy:

1) According to the TSH circulating levels measured in different TR knockouts, TRß1 and TRß2 cooperate for down-regulation. Two studies describe the knockout of only TRß2. Although the increase in TSH level varies between the two studies, both lead to the conclusion that TRß2 is the main isoform regulating TSH and TSHß genes in pituitary thyrotrope cells (98, 101). In the hypothalamus, TRß2 is the main regulator of the TRH gene (129). The very high levels of TSH in animals lacking both THRA and THRB (105, 130) argue against the requirement of unliganded TR for activating TSH and TSHß gene expression. It also indicates that TR1 can partially compensate for the absence of TRß1 and TRß2 in THRB knockout animals.

2) Administration of the RXR synthetic agonist AGN194204 results in an equivalent decrease of TSH secretion in both wild-type and THRB-deficient mice. This effect is thus unlikely to be mediated by RXR/TRß heterodimers, and the intervention of RXR in TRß mediated down-regulation of TSH genes remains an open question (131).

3) A mutation that prevents DNA binding by TRß1 and TRß2 (G125S) abrogates down-regulation of TSH genes (132). Whether DNA binding must occur on negative TREs for TSH genes to be down-regulated remains to be shown.

4) Coactivator recruitment by TRß2 is required for TSH down-regulation. This is demonstrated by the consequence of the highly selective E457A mutation, which prevents coactivator recruitment without modifying corepressor binding and causes dysregulation of TSH genes in knock-in mice (133). This conclusion is consistent with some genetic (134) and in vitro (135) observations but, again, does not demonstrate the direct involvement of a negative TRE.

5) Eliminating the genes encoding the histone acetylase transferase coactivators, SRC1 and, to a lesser extent, TIF2, results in an increase in TSH expression (136, 137).

Taken together, these observations lead to the astonishing proposal that T₃-dependent repression results from the recruitment of histone acetylase on TSH gene promoters, perhaps via negative response elements. A precise definition of the histone code in these regions might provide an explanation to this paradox. Although suggestive, these genetic data should be interpreted with caution, taking into account two difficulties. First, the THRB mutations usually affect the secretion of both TSH and TRH. Because TRH is absolutely required for TSH synthesis, as demonstrated by the gene knockout (138), THRB mutations act both directly and indirectly on TSH secretion. Second, THs regulate not only TSH secretion but also pituitary development. Congenital hypothyroidism, as observed in Pax8^–/– mice, induces thyrotrope cell hypertrophy and hyperplasia, at the expense of somatotropes and lactotropes. These defects cannot be fully corrected by late T₃ treatment (139). Furthermore, one of the THRB knock-in mutations was found to promote pituitary tumor formation (80). These difficulties outline the urgent need for experimental models allowing for a spatio-temporal control of genetic mutations.

	FUTURE DIRECTIONS: NEW ANIMAL MODELS, NEW LIGANDS

TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 
From the previous overview, it is tempting to speculate that T₃ signaling results from the evolution of an ancestral situation in which TH and other iodinated compounds were already present, acting without binding to TR. TR then evolved to bind one of these iodinated molecules, T₃, which became able to modulate its transcriptional activity (140). After genome duplications, occurring before the appearance of vertebrate, THRA and THRB functions diverged. In most tissues, TR1 seems to be the most active isoform in the absence of ligand in repressing gene expression, and in priming the T₃ response during embryogenesis or cell differentiation. Although T₃ self-induction, which is observed in amphibian metamorphosis, is not conserved for the mammalian THRB gene, TRß1 often seems to act as a natural amplifier of T₃ response. This is the case in the heart (141). This hypothetical scenario leads to testable hypotheses for further investigations. As our understanding of transcriptional mechanisms progresses, it is likely that new possibilities for TH and TR modes of action will be proposed, because it appears that TRs participate in a large network of protein-protein interactions. In this complex situation, it is of utmost importance that complementary genetic evidence is provided to confirm the relevance of molecular mechanisms in physiological or pathological settings. For example, viable mice devoid of all TRs are ideal to address the implications of TR-independent action of TH, and the cross talk between TR and other nuclear receptors signaling pathways could be studied in mice carrying a point mutation preventing the DNA binding of one TR. Current studies often face the difficulty of separating the various functions of each TR in vivo. For example, behavioral abnormalities observed in THRA knock-in animals relate to both developmental impairments and adult homeostatic dysregulation (95). CRE/loxP-mediated recombination in transgenic mice is currently the favorite tool in addressing these challenges. Another problem that will require advanced transgenic technology is the understanding of negative regulation exerted by liganded TRß on the TRH, TSH, and TSHß genes. New germline or somatic mutations eliminating the negative TREs from promoters, or abrogating key interactions between TRß and cofactors, will be required.

Human genetic studies have focused on the RTH syndrome, which is usually due to THRB point mutations occurring in one the three hot spots in the reading frame of the ligand-binding domain (142). To date, no germline mutation in the human THRA gene has been reported. However, it is anticipated from mouse studies that the phenotype resulting from such mutations, if they exist, will be very different than that of RTH. For example body growth, lipid metabolism, glycemia, and cognitive functions could be impaired. In some RTH patients, neither THRB nor THRA is mutated, leaving the possibility that a gene crucial for TRß function remains to be identified. Mouse genetic studies (79, 85, 143, 144, 145) and sporadic observations in humans (146, 147, 148) also suggest that TR somatic mutations participate in the development of various tumors. The association between THRA and cancer has been predicted for a long time, due to its affiliation with the v-erbA, an avian retrovirus oncogene that acts in a dominant-negative manner on TR signaling (149).

Pharmacological modulation of TR functions should be complementary to experimental genetic studies. However, up to now, very few synthetic TR ligands have been developed. TR agonists or antagonists would be of great interest if they could act in an isotype-, isoform-, or tissue-specific manner. GC-1 and KB-141 are two closely related compounds, which hold promise, because they can act as selective TRß agonists. In animal models, they decrease plasma cholesterol and triglycerides levels and induce fat loss, without exerting significant effects on heart and muscle. Fortunately, and unexpectedly, these compounds have a moderate effect on TSH secretion and do not block endogenous TH production (150). Also unexplained is the fact that GC-1 acts differently within cardiomyocytes on different response elements (151) The more recently designed NH3 compound acts as a relatively selective antagonist, placing the TR1 receptor in a neutral conformation that does not permit either coactivator or corepressor recruitment (152, 153). Other ligands, discovered by high throughput wet screens or virtual screening, are currently being evaluated (154, 155, 156, 157, 158, 159, 160).

Surprisingly, the various THRA and THRB knock-in mutations have different effects on mouse development and homeostasis. For example, only one of the THRA mutations causes obesity, and only one of the THRB mutations affects cerebellar development. Although other explanations cannot be excluded, an attractive possibility would be that this phenotypic diversity mirrors various receptor conformations, stabilized by the amino acid changes. If this hypothesis is true, future synthetic ligands should be able to place TR in several different conformations with different physiological outcomes and act as STORMs. Once available, these new drugs will probably have extensive therapeutic applications.

	ACKNOWLEDGMENTS

 
We thank Paul Yen and Andy Shulman for critical reading of the manuscript and helpful discussion.

	FOOTNOTES

 
Research in our laboratory was supported by the French Ministery of Research (ACI Biologie Cellulaire, Moléculaire et Structurale) Ligue contre le Cancer (équipe labelisée), and the CASCADE European Network of Excellence (European Union Contract No. FOOD-CT-2004-506319).

The authors have nothing to declare.

First Published Online June 8, 2006

Abbreviations: AP1, Activator protein 1; CRC, chromatin-coupling complex; CREB, cAMP-responsive element binding protein; LXR, liver X receptor; OATP, organic anion transporting polypeptide; PI3K, phosphatidylinositol-OH-3-kinase; PPAR, peroxisome proliferator-activated receptor; RTH, resistance to thyroid hormone; RXR, retinoic X receptor; STORM, selective TR modulator; TBL1, transducin-like 1; TH, thyroid hormone; TR, TH receptor; TRE, T₃ response element.

Received for publication January 20, 2006. Accepted for publication May 31, 2006.

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TOP ABSTRACT INTRODUCTION TRs ARE LIGAND-REGULATED... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... OTHER LEVELS OF REGULATION:... CROSS TALK: TR FUNCTIONS... RESPECTIVE FUNCTIONS OF TH... DO UNLIGANDED RECEPTORS REGULATE... UPSIDE-DOWN-REGULATION: TSH DOWN... FUTURE DIRECTIONS: NEW ANIMAL... REFERENCES

 

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