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Endocrinology, doi:10.1210/en.2003-1209
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Endocrinology Vol. 145, No. 5 2337-2345
Copyright © 2004 by The Endocrine Society

Both Thyroid Hormone Receptor (TR)ß1 and TRß2 Isoforms Contribute to the Regulation of Hypothalamic Thyrotropin-Releasing Hormone

Sandrine M. Dupré, Hajer Guissouma, Frédéric Flamant, Isabelle Seugnet, Thomas S. Scanlan, John D. Baxter, Jacques Samarut, Barbara A. Demeneix and Nathalie Becker

Muséum National d’Histoire Naturelle (S.M.D., H.G., I.S., B.A.D., N.B.), Unité Scientifique du Muséum 501 Département Régulation, Développement et Diversité Moléculaire, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 5166, 75231 Paris Cedex 05, France; Laboratoire de Biologie Moléculaire et Cellulaire de l’Ecole Normale Supérieure de Lyon (F.F., J.S.), CNRS, UMR 5665 Laboratoire Associé Institut National de la Recherche Agronomique 913, 69364 Lyon Cedex 07, France; and the Metabolic Research Unit, Department of Pharmaceutical Chemistry and Molecular and Cellular Pharmacology (T.S.S., J.D.B.), University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Dr. Barbara A. Demeneix or Dr. Nathalie Becker, Muséum National d’Histoire Naturelle, Unité Scientifique du Muséum 501 Département Régulation, Développement et Diversité Moléculaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5166, 75231 Paris Cedex 05, France. E-mail: demeneix{at}mnhn.fr or becker{at}mnhn.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormones (TH) are essential regulators of vertebrate development and metabolism. Central mechanisms governing their production have evolved, with the ß-TH receptor (TRß) playing a key regulatory role in the negative feedback effects of circulating TH levels on production of hypothalamic TRH and hypophyseal TSH. Both TRß-isoforms (TRß1 and TRß2) are expressed in the hypothalamus and pituitary. However, their respective roles in TH-dependent transcriptional regulation of TRH are undefined. We confirmed the preferential role of TRß vs. TR{alpha} isoforms in TRH regulation in wild-type mice in vivo by using the TRß preferential agonist GC-1. We next determined the effects of tissue-specific rescue of TRß1 and TRß2 isoforms by somatic gene transfer in hypothalami of TRß null (TRß–/–) mice. TH-dependent TRH transcriptional repression was impaired in TRß–/– mice, but was restored by cotransfection of either TRß1 or TRß2 into the hypothalamus. TRß1, but not TRß2, displayed a role in ligand-independent activation. In situ hybridization was used to examine endogenous TRH expression in the paraventricular nucleus of the hypothalamus of TRß–/– or TR{alpha} null (TR{alpha}o/o) mice under different thyroid states. In contrast to published data on TRß2–/– mice, we found that both ligand-independent TRH activation and ligand-dependent TRH repression were severely impaired in TRß–/– mice. This study thus provides functional in vivo data showing that both TRß1 and TRß2 isoforms have specific roles in regulating TRH transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BECAUSE THYROID HORMONES (TH) play important roles in vertebrate metabolism and development, levels of free TH in blood must be finely regulated. This control relies on positive inputs and negative feedback elements along the hypothalamo-pituitary thyroid gland axis: TRH synthesized in the hypothalamus, stimulates TSH production in the anterior pituitary, secretion of which induces TH synthesis by the thyroid gland. In return, increasing levels of free TH in blood repress TRH and TSH expression by negative feedback. However, the molecular mechanisms underlying these essential negative regulatory components are poorly understood.

The most active form of TH, T3, acts via TH receptors (TRs), which belong to the nuclear receptor class II. These receptors have ligand-dependent and -independent actions (1). TRs are encoded by TR{alpha} (NR1A1) and TRß (NR1A2) genes (2, 3, 4), which produce eight variants: TR{alpha}1, TR{alpha}2, TR{Delta}{alpha}1, TR{Delta}{alpha}2 (2, 3, 5, 6, 7, 8) and TRß1, TRß2, TRß3, TR{Delta}ß3 (9, 10, 11, 12, 13). Among these isoforms, only TR{alpha}1, TRß1, TRß2, and TRß3 can bind both T3 and TH response element (TRE) DNA binding sites. Such TRs can bind to TREs as monomers, homodimers, or heterodimers with retinoid X receptor (14, 15). Succinctly, when bound to a positive TRE, unliganded TRs recruit a multiple protein complex, including nuclear corepressors. Binding of T3 induces conformational change of TRs, leading to corepressor release and coactivator recruitment. By these mechanisms transcription of positively regulated genes via positive TREs is repressed in the absence of ligand and activated in its presence.

However, despite much accumulated data that have provided working models of TR action on positive TREs, the mechanisms of TR action on negative TREs such as those found in the TRH and TSH genes remain unclear. We focused our attention on TRH regulation by TRs in the paraventricular nucleus of the hypothalamus (PVN), because this region is critical in controlling thyroid function via negative feedback by T3 (16, 17, 18, 19). Previous work employing TRH promoter constructs in hypothalamic cultures and in mice hypothalami demonstrated that this T3-dependent negative regulation occurs at the transcriptional level (20, 21, 22). Furthermore, both in vitro (20, 23, 24) and in vivo (21, 25) studies showed that TRß isoforms, but not TR{alpha} isoforms, are capable of mediating ligand-dependent repression of TRH. Moreover, the N terminus of TRß was shown to be the key element in this regulation (22). A central question remaining is the potential contribution of the different TR isoforms to the transcriptional TRH regulation.

To understand the implication of TR isoforms in T3-dependent pathways, different TR knockout mice have been created (for reviews, see Refs. 26, 27, 28). Only two studies have adressed TRH regulation in mutant mice. In situ hybridization (ISH) data showed that TRß2 deletion alone, or deletion of both TRß1 and TRß2 isoforms, impaired TRH repression during hyperthyroidism (25, 29). Here, we chose to examine TRH regulation, in both hypothyroid and hyperthyroid states, in the hypothalamus of mice lacking both TRß1 and TRß2 isoforms [TRß–/– (30)] or lacking TR{alpha}1, {alpha}2, {Delta}{alpha}1 and {Delta}{alpha}2 isoforms [TR{alpha}o/o (31)]. Using ISH we found that TRH regulation was severely impaired in TRß–/– mice, with ligand-independent activation being noticeably abrogated compared with wild-type and TRß2–/– animals (25), underlining a role for TRß1 in TRHregulation. In vivo transfection assays showed that both TRß1 and TRß2 could restore lost ligand-dependent regulation of transcription in TRß–/– mice. We conclude that both TRß isoforms contribute to regulation of TRH mRNA transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
TRH-luciferase (TRH-luc), rTRß1 and rTR{alpha}1 constructs were described by Guissouma et al. (22). rTRß2 cDNA was obtained from V. Nikodem (National Institutes of Health, Bethesda, MD) and subcloned into pSG5 (Stratagene, La Jolla, CA).

Animals
TRß–/– mice (30) lacking TRß1 and TRß2 were maintained on a 129/Sv background. TR{alpha}o/o mice (31) lacking TR{alpha}1, TR{alpha}2, TR{Delta}{alpha}1, and TR{Delta}{alpha}2, were maintained on a C57BL/6J background. Wild-type 129/Sv (TRß+/+) and C57BL/6J (TR{alpha}o/o) were purchased from Charles River Laboratories (l’Arbresle, France). Swiss wild-type mice were purchased from Janvier (Le Genest St Isle, France). All aspects of animal care and experimentation performed in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Protection et Santé Animale, Direction des Services Vétérinaires de Paris (Animal Protection and Health, Veterinary Services Direction, Paris, France).

In vivo transfection and luciferase assays
To induce fetal and neonatal hypothyroidism, dams were given an iodine-deficient food containing 0.15% 6-n-propyl-2-thiouracil (PTU) (Harlan, Gannat, France) on d 14 of gestation. This diet was continued throughout the lactation period. DNA/PEI (polyethylenimine) complexes and in vivo transfections were carried out as previously described (21). Pups were anesthetized by hypothermia on ice and injected on postnatal d 1. A glass micropipette was lowered 2 mm through the skull, approximately 1 mm lateral to the sagittal suture, into the hypothalamic area. Two microliters of a 5% glucose solution containing plasmid/PEI complexes were slowly injected bilaterally. Then, for evaluating T3 (Sigma-Aldrich, St Quentin Fallavier, France) or GC-1 (32) effects on reporter gene expression, hypothyroid pups were injected sc with 250 µg of T3 (or 122 µg of GC-1, respectively) per 100 g of body weight (b.w.) in 0.9% saline, corresponding to 370 nmol/100 g b.w. of T3 or GC-1. Ligand effects were also evaluated at 37 nmol/100 g b.w. Controls received the same volume of 0.9% saline. After T3 treatment, levels of total circulating T3 were above 690 ng/dl for each group. After 18 h, mice were decapitated; the hypothalami were dissected out for luciferase analyses. TRH-luc plasmid was a gift from W. Balkan (University of Miami School of Medicine, Miami, FL); its use is described in Becker et al. (33). Given the highly tissue-specific nature of TRH transcription, one of the most important steps in ensuring reproducibility is careful and consistent microdissection of the hypothalamic areas transfected (21).

ISH
Hypothyroidism in adult (4 month old) mice was induced by giving an iodine-deficient food containing 0.15% PTU (Harlan) for 1 month. Hyperthyroidism was induced by giving T3 (Sigma-Aldrich) at 1.5 µM in the drinking water for 1 month. T3 and not T4 is given to the animals because it is known that T3 is able to pass the brain-blood barrier (34, 35), and T3 is the most active form of the molecule.

Vibratome brain sections were processed as adapted from Wilkinson (36) and as previously described (33). A cDNA insert of the mouse prepro-TRH gene (gift from M. Yamada, School of Medicine, Maebashi, Japan) was used for the synthesis of labeled probes. The sense probe did not provide any detectable signal (33).

Measurement of total plasma T3
Frozen plasma was thawed and processed according to the supplier’s instructions, using the AMERLEX-M T3 RIA Kit (Trinity Biotech, Wicklow, Ireland). Results are expressed as means ± SEM.

Statistical analysis of the results
In vivo gene transfer results are expressed as mean ± SEM per group. After ANOVA analysis where appropriate, the Wilcoxon test was used to analyze differences between groups. Differences were considered significant at P <= 0.05. Each experiment was carried out with n >= 10 mice per group and repeated at least three times. The low fertility of the 129/Sv strain resulted in small broods of four to six pups. Thus, when following TRH-luc transcriptional regulation in TRß–/– mice and their wild-type controls, we combined results from different experiments. Because basal, control levels of TRH-luc from one series of experiments to another could differ, the results for each treated group were normalized against the mean luciferase values from their respective internal controls.

For ISH, TRH-positive cells in the PVN and in the perifornical area (PFA) were counted individually by two persons, each counting four to six half-sections for each animal. An average of four animals per group was used, and results were expressed as means ± SEM for PVN and PFA (see Fig. 3Go, C and D). Because the results showed that TRH expression in the PFA is unaffected by the thyroid status of the animals, the number of labeled cells in the PVN was normalized against the number of TRH-expressing cells in the PFA for each half-section (see Fig. 4Go, C and F). Thus, TRH mRNA levels are expressed as PVN/PFA ratios, given as means ± SEM. Wilcoxon’s test was then used to analyze the difference between groups. Differences were considered significant at P <= 0.05.



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  		FIG. 3. ISH shows TRH expression to be regulated by T3 in the PVN but not in the PFA of the hypothalamus. Coronal vibratome sections (100 µm) of brains from adult wild-type (129/Sv) male mice were hybridized with a digoxigenin-labeled antisense TRH RNA probe and revealed with alkaline phosphatase conjugated antidigoxigenin antibody. The left panel displays a typical view of the area surrounding one hypothalamic PVN, in mice brain slices after ISH. Wild-type mice were treated with PTU (A) or T3 (B), respectively. F, Fornix; III V, third ventricle. C and D, The data represent the number of TRH-labeled cells in the PVN (C) and in the PFA (D), in PTU-, untreated, or T3-treated mice. Means ± SEM are given. Values were obtained from four to six sections, from an average of four brains per group. ***, P < 0.001; n.s., not significant. Scale bars represent 100 µm.

 


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  		FIG. 4. Both ligand-independent activation and ligand-dependent repression of TRH expression are impaired in the PVN of TRß–/– mice hypothalami. On the contrary, physiological regulation of TRH is amplified in TR{alpha}o/o mice. Coronal vibratome sections (100 µm) of brains from adult TRß–/– male mice were hybridized with a digoxigenin-labeled antisense TRH RNA probe and revealed with alkaline phosphatase conjugated antidigoxigenin antibody. A, B, D, and E display typical views of the area surrounding one hypothalamic PVN, in mice brain slices after ISH. TRß–/– and TR{alpha}o/o mice were treated with PTU (A and D) or T3 (B and E), respectively. F, Fornix; III V, third ventricle. C and F, labeled cells were counted and normalized for each PVN against the number of labeled cells in the PFA, in PTU-, untreated, or T3-treated mice. White columns, Wild-type 129/Sv mice (C) or C57BL/6J mice (F). Hatched columns, TRß–/– mice (C) or TR{alpha}o/o mice (F). Values are from four to six PVN/PFA counted per brain, from an average of four brains per group. Means ± SEM are given. *, P < 0,05; **, P < 0.01; ***, P < 0.001; n.s., not significant. Scale bars represent 100 µm.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Use of GC-1, a TRß preferential agonist, confirms the key role of TRß isoforms in TRH regulation
To obtain functional data on the contributions of the different TR isoforms to T3-dependent repression of the TRH gene, we used a transfection assay with a TRH-luc construct. TRH-luc transcription was followed in vivo directly in the hypothalamus of newborn wild-type mice. To analyze the specific contributions of type {alpha} vs. type ß TR isoforms on TRH-luc regulation, we treated the animals either with T3, or with GC-1, a TRß-preferential agonist with a similar affinity to T3 (32). T3 was used at amounts equivalent to those known to induce a T3-dependent transcriptional repression in typical experiments (2.5 µg/g of b.w.), and GC-1 was used at equimolar amounts. As shown previously (21, 22), expression from TRH-luc was significantly reduced (23%; P < 0.01) in animals injected with 370 nmol/100 g b.w. T3. A similar reduction (29%; P < 0.05) was observed when treating the animals with equimolar amounts of GC-1 (Fig. 1Go). To verify GC-1 specificity, doses were reduced 10-fold. T3-dependent repression of TRH-luc was lost at this dosage, whereas GC-1 still induced a ligand-specific repression (37%; P < 0.001; Fig. 1Go). Thus, at equimolar doses, GC-1 is more effective than T3 in inducing a ligand-dependent transcriptional repression of TRH-luc. These results suggest that the liganded TRß isoforms are sufficient to confer a ligand-dependent transcriptional repression of TRH-luc.



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  		FIG. 1. Repression of TRH-luc by the TRß preferential agonist GC-1 in wild-type mice. TRH-luc expression was measured in hypothyroid 1-d-old mice (see Materials and Methods) treated with T3, GC-1 or vehicle (ct), 18 h after hypothalamic injection of 1 µg TRH-luc reporter construct. GC-1 was injected at an equimolar dose to T3, corresponding to 2.5 µg T3/g of b.w. (black columns), or at a 1/10th dilution corresponding to 0.25 µg T3/g of b.w. (gray columns). The controls received vehicle injections (ct, white column). Means ± SEM are given, n >= 10 per point. The whole experiment was repeated three times with similar results. *, P < 0.05; ***, P < 0.001; n.s., not significant.

 
Specific contributions of TRß1 and TRß2 isoforms to T3-dependent repression of a TRH-luc transgene in vivo
To evaluate the contributions of TRß1 and TRß2 to T3-dependent regulation of TRH-luc, we used TRß knockout mice, devoid of any ß-type isoform (30) rendered hypothyroid by PTU treatment. After PTU treatment, circulating total T3 levels of TRß–/– newborn mice were significantly reduced [from 34.2 ± 3 ng/dl (n = 8) to 25.2 ± 1.4 ng/dl (n = 5); P < 0.05]. Thus, T3 levels of PTU-treated TRß–/– newborn mice were similar to those in their wild-type, PTU-treated counterparts [25.2 ± 1.4 ng/dl (n = 5) vs. 29.3 ng/dl ± 1.3 (n = 9), respectively].

Using the in vivo transfection assay, we first checked that T3-dependent transcriptional repression of TRH-luc was present in 129/Sv pups, the wild-type controls for TRß–/– mice. We found a significant T3-dependent reduction (–36%; P < 0.05) of TRH-luc expression in wild-type pups (Fig. 2AGo). The same protocol was applied to newborn TRß–/–: no regulation of TRH-luc by T3 was observed (Fig. 2BGo, first pair of columns). These results confirm that TRß isoforms are essential to this T3-dependent mechanism. Cotransfection of TRß1 rescued this ligand-dependent regulation, as did cotransfection of TRß2 (Fig. 2BGo, second and third pairs of columns). TRß1 and TRß2 provided 34% (P < 0.05) and 39% (P < 0.001) repression, respectively, in the presence of T3 vs. saline. Thus, both ß1 and ß2 TR isoforms are able to restore the T3-dependent transcriptional repression of TRH-luc in TRß–/– mice. In the absence of T3, moreover, TRß1 promoted a small (22%) but significant (P = 0.05) T3-independent transcriptional activation of TRH-luc, compared with controls (Fig. 2BGo, compare first and second white columns), whereas TRß2 did not. In conclusion, both TRß1 and TRß2 isoforms mediate a T3-dependent transcriptional repression of TRH-luc, whereas TRß1 alone contributes to T3-independent transcriptional activation of TRH-luc.



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  		FIG. 2. A, TRH-luc regulation by T3 in TRß+/+ mice. TRH-luc expression was measured in hypothyroid 1-d-old mice (see Materials and Methods) treated with T3 (2.5 µg/g of b.w., +T3) or saline (–T3), 18 h after hypothalamic injection of 1 µg TRH-luc reporter construct. Values were normalized with the mean obtained with saline. The whole experiment was repeated three times with similar results. Means ± SEM are given, n >= 10 per point. *, P < 0.05. B, Cotransfection of TRß1 or TRß2 restores TRH-luc regulation by T3 in TRß–/– mice. TRß1 mediates a T3-independent TRH-luc activation. TRH-luc expression was measured in hypothyroid TRß–/– 1-d-old mice (see Materials and Methods) treated with T3 (2.5 µg/g of b.w., +T3) or saline (-T3), 18 h after hypothalamic injection of 1 µg TRH-luc and 100 ng of expression vector [control pSG5 (ct), pSG5-TRß1 (TRß1), or pSG5-TRß2 (TRß2)]. Values were normalized with the mean obtained with saline. Means ± SEM are given, n >= 10 per point. The whole experiment was repeated three times with similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

 
Quantification of endogenous TRH mRNA expression in the hypothalamus
To investigate the effects of TRß or TR{alpha} ablation on expression of endogenous TRH in different thyroid conditions, we used nonradioactive ISH. First, wild-type controls (129/Sv for TRß–/– mice, or C57BL/6J for TR{alpha}o/o mice) were treated with either PTU or T3, before applying ISH to brain sections. Examining the anterior hypothalamic region of 129/Sv mice revealed two distinct TRH-positive regions: the PVN and PFA (Fig. 3Go, left panel). In the PVN, labeling was particularly intense in hypothyroid animals, whereas it was barely detectable in hyperthyroid animals (Fig. 3Go, left panel, compare A with B). On the contrary, expression in the PFA was not modified, whatever the thyroid status of the animal. To assess the ISH signal quantitatively, the labeled cells of each section were counted (see Materials and Methods) in the PVN and PFA, respectively (Fig. 3Go, C and D). As already described for the rat hypothalamus (19), TRH mRNA expression is regulated by the free TH in the blood of the animal, specifically in the PVN. Indeed, we found that PTU treatment induced a significant up-regulation of TRH mRNA expression in the mouse PVN (P < 0.001), whereas the numbers of labeled cells remained unchanged in the PFA (Fig. 3Go, C and D). In contrast, in the PVN, numbers of labeled cells were reduced 10-fold after T3 treatment (P < 0.001) (Fig. 3CGo). This was again specific to the PVN because no variation was seen in the PFA (Fig. 3DGo). Results were similar for C57BL/6J mice (data not shown). Thus, normalizing labeled cell numbers in PVN vs. PFA after ISH permits quantification of endogenous TRH mRNA transcription in mice PVN under different thyroid states.

Loss of TRß isoforms abolishes physiological TRH regulation
To examine the effects of TRß ablation on the expression of endogenous TRH mRNA, we first compared TRß–/– knockout mice with their wild-type 129/Sv counterparts. Brain sections were processed for ISH and TRH mRNA expression monitored in the PVN, using PFA labeling as an internal control. In wild-type controls, normalization of positive cell numbers in the PVN vs. numbers in the PFA showed TRH mRNA expression to be doubled in PTU-treated animals, and reduced to 35% of controls in T3-treated animals (in each case, P < 0.001; Fig. 4CGo; compare white columns). Similar results have been obtained using wild-type mice and radioactive ISH (25).

In TRß–/– mice, there was no obvious change in hypothalamic TRH mRNA distribution (Fig. 4Go, A and B). However, there was a significant reduction in TRH mRNA-positive cells in the PVN of TRß–/– mice compared with their wild-type counterparts (–36%; P < 0.05, Fig. 4CGo; second pair of columns). In these two groups of untreated mice, T3 levels were not significantly different [59.9 ± 4.3 ng/dl (n = 13) in TRß–/– mice vs. 54.2 ± 2.3 ng/dl (n = 11) in wild-type mice]. These T3 values differ somewhat from the findings of others (30, 37, 38) who reported higher levels of T3 in 3- to 10-wk-old TRß–/– mice; however, the mice we used in these experiments were older (16 wk). It has been shown that the difference in T4 levels between wild-type mice and TRß–/– mice significantly decrease with age (38). PTU treatments of TRß–/– mice significantly reduced T3 levels [20.6 ± 3.5 ng/dl (n = 2)], as did PTU treatment on wild-type controls [13.1 ± 5.1 ng/dl (n = 3)]. In PTU-treated TRß–/– mice, TRH mRNA expression was again significantly lower than in wild-type hypothyroid controls (compare Fig. 4AGo with Fig. 3AGo; see Fig. 4CGo, first pair of columns). Thus, our results show that ablation of both TRß1 and TRß2 isoforms abrogates the physiological up-regulation of TRH mRNA expression in the absence of T3.

In contrast, in TRß–/– mice rendered severely hyperthyroid by T3 treatment [969.6 ± 379.9 ng/dl (n = 6)], there was no repression of TRH mRNA. In fact, TRH mRNA expression was significantly higher (Fig. 4CGo, third pair of columns; P < 0.001) than in hyperthyroid wild-type mice, which had T3 values of 746.5 ± 280.1 ng/dl (n = 5). Moreover, these levels of TRH mRNA expression in hyperthyroid TRß–/– mice were significantly greater than in PTU-treated (P < 0.05) or untreated (P < 0.01) TRß–/– mice (Fig. 4Go, compare left panels A and B; right panel, compare three shaded columns). Thus, absence of TRß1 and TRß2 not only abrogates T3-dependent transcriptional repression of the TRH gene, but also actually results in T3-dependent increased levels of TRH.

Loss of TR{alpha} isoforms strengthens physiological TRH regulation
To examine the effects of TR{alpha} ablation on endogenous TRH in the PVN, we modulated the thyroid status of wild-type controls (C57BL/6J) and TR{alpha}o/o mice (31). Total T3 levels were again measured for each treatment, in wild-type and TR{alpha}o/o mice, respectively: 19.9 ± 5.2 ng/dl (n = 3) and 17.7 ± 3.1 ng/dl (n = 2) in hypothyroid mice, 45.9 ± 4.2 ng/dl (n = 5) and 66.1 ± 6.3 ng/dl (n = 3) in untreated mice, 269.9 ± 109.5 ng/dl (n = 3) and 214.2 ± 66.2 ng/dl (n = 3) in hyperthyroid mice. We found no significant difference in T3 levels between wild-type and TR{alpha}o/o mice in each group. Endogenous TRH mRNA expression was analyzed by ISH as described for the TRß–/– mice. Again, we observed a significant up-regulation of TRH in hypothyroid C57BL/6J controls (+43%; P < 0.05), and a significant T3-dependent transcriptional down-regulation of TRH (–45%; P < 0.001) in hyperthyroid controls, compared with untreated, euthyroid controls (Fig. 4FGo, white columns). At the histological level, we observed a T3-dependent repression of TRH mRNA expression in the PVN of hyperthyroid TR{alpha}o/o mice (Fig. 4Go, compare D and E). Quantification showed that PTU treatment induced a significant up-regulation (+48%; P < 0.05, compared with untreated TR{alpha}o/o mice) of TRH expression in the PVN, and T3 treatment resulted in a down-regulation (–76%; P < 0.001, compared with untreated TR{alpha}o/o mice) of the TRH gene (Fig. 4FGo, compare the three shaded columns). Thus, physiological TRH regulation is maintained in TR{alpha}o/o mice. However, the extent of TRH regulation in different thyroidal states was amplified in TR{alpha}o/o knockout mice compared with wild-type control mice. First, PTU treatment resulted in a stronger increase of the number of neurons expressing TRH mRNA in TR{alpha}o/o mice than in wild-type mice (Fig. 4FGo, first pair of columns; P < 0.05). Second, T3 treatment led to a greater down-regulation of TRH mRNA expression in TR{alpha}o/o mice than in wild-type control mice (Fig. 4FGo, third pair of columns; P < 0.001). We conclude that physiological regulation of TRH is amplified in TR{alpha}o/o mice, where only TRß isoforms can enter into play.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides the first functional in vivo data on the specific roles of TRß1 and ß2 isoforms in regulating TRH transcription. The approach applied exploits two powerful techniques: first, the analysis of the consequences of the removal of the TRß gene by homologous recombination in mice, and, second, the tissue-specific rescue of TRß1 and TRß2 isoforms by somatic gene transfer in hypothalami of TRß–/– mutant mice. These data are novel in that they are the first to provide data on rescue of TRß1 and TRß2 isoforms in the hypothalamus of mutant mice; and, second, they show the specific effect of the lack of TRß isoforms on endogenous TRH expression under different thyroid states.

TRH regulation by TH in the PVN is the central set point controlling circulating TH levels. Immunocytochemical studies have demonstrated that four TR isoforms are found in the PVN: TR{alpha}1, TR{alpha}2, TRß1, and TRß2 (39, 40, 41). Each of these isoforms, except TR{alpha}2, binds T3. In keeping with the marked expression of TRß2 mRNA in the hypothalamic PVN and in the pituitary, this particular TR was shown to be the main isoform responsible for the T3-dependent regulation of the TRH gene. Indeed, the repression of TRH by T3 was shown to be impaired in the PVN of TRß2–/– mice (25). However, a number of functional studies in wild-type mice hypothalami in vivo (21, 22) and in vitro (23) have shown that TRß1, as well as TRß2, overexpression is compatible with ligand-dependent TRH repression, whereas TR{alpha}1 overexpression blocks physiological regulation of TRH transcription. These results therefore raise the question of the precise contributions of TRß1 and TRß2 to transcriptional regulation of the TRH gene.

To dissect the transcriptional effects of the different TR isoforms in vivo, we used an in vivo gene transfer method. This method involves the introduction of a plasmid construct containing a TRH promoter sequence cloned upstream of the luciferase reporter gene (TRH-luc). The physiological relevance of the assay has been demonstrated previously, and transcription of the TRH-luc construct being significantly up- and down-regulated when transfected into hypothalami of hypothyroid or hyperthyroid mice, respectively (21, 22), the regulations observed being parallel to that of endogenous TRH (19). Although in the same order of magnitude, these regulations were not as ample as those seen for endogenous TRHin adult animals. These differences may be due to four factors, which vary between gene transfer experiments on newborns and ISH on adults: 1) the region examined (whole hypothalamic region vs. PVN); 2) the duration and administration of T3 (single sc injection vs. 1 month of oral administration); 3) the levels of endogenous TRHexpression; and 4) the detected molecule (luciferase vs. mRNA).

As stated above, the key result from these first in vivo gene transfer experiments was the identification of a physiological role for TRß, but not TR{alpha}, isoforms in TRH regulation. An important step in furthering these analyses was to exploit a recently synthesized TRß-preferential ligand, GC-1 (32). Given that this ligand will induce transcriptional effects on TR-responsive genes by binding preferentially to TRß, we tested its effects on TRH regulation in wild-type mice. Although GC-1 availability in the brain cannot be assessed, we observed ligand-dependent TRH-luc repression, similar in amplitude to that induced by injection of equivalent amounts of T3. Moreover, at a lower dose (37 nmol/100 g b.w.), GC-1 was even more potent in the ligand-dependent transcriptional repression of TRH-luc. At this dose, GC-1 probably has a better selectivity for TRß (42). These data thus confirm the predominant role of the ß-type isoforms, as well as the physiological pertinence of this assay. In particular, the fact that the lower dose of GC-1 (possibly more ß-selective) was more effective also suggests that the liganded TR{alpha} isoforms do not contribute to (or even interfere with) ligand-dependent transcriptional repression of TRH-luc.

In the following series of experiments, we applied in vivo somatic gene transfer approach to TRß–/– knockout mice. No T3-dependent transcriptional regulation of TRH-luc was seen in these mice (Fig. 2BGo). Rescuing either hypothalamic TRß1 or TRß2 isoform expression by in vivo gene transfer restored T3-dependent transcriptional repression. The degree of ligand-dependent transcriptional repression was equivalent with each TRß isoform and was not different from levels seen in control, wild-type mice. This functional data allows us to conclude that both TRß1 and TRß2 isoforms can mediate T3-dependent transcriptional repression of the TRH gene. However, ISH data on TRH mRNA expression in TRß2–/– mutant mice (25), lacking TRß2 but not TRß1, do not fully corroborate this conclusion. In the TRß2–/– mutant mice, T3-dependent transcriptional repression of hypothalamic TRH is lost. A possibility is that the levels of TRß1 receptors in the PVN are not sufficient to compensate for the loss of TRß2 in T3-dependent repression. This raises the question of the effect of TRß2 ablation on TRß1 expression in the hypothalamus, a possibility that has not been investigated.

One additional result arising from the rescue studies reported here was the TRß1 isoform-specific, T3-independent transcriptional activation of TRH-luc. This result is corroborated by comparing results from ISH studies of TRH mRNA expression in TRß–/– mice (this study) and TRß2–/– mutant mice (25) (Fig. 5Go). In mice lacking only the TRß2 isoform, TRH mRNA expression is maintained at a maximal level, roughly equivalent to hypothyroid wild-type mice whatever the thyroid state of the mutant mice (25). These elevated TRH mRNA levels suggest that the presence of TRß1, in the absence of T3 and TRß2, has an activation function. Indeed, in hypothyroid mice lacking both TRß isoforms, TRHexpression is repressed at levels beyond those of euthyroid wild-type mice, and far beyond those of TRß2–/– mutant mice (Fig. 5Go, first two pairs of columns). This finding indicates that lack of TRß1 results in a reduction of the T3-independent activation of TRH. This role of TRß1 in activation of T3-independent TRHwas not revealed in the recent study of Abel et al. (29) on TRß–/– mice, as these authors concentrated on the hyperthyroid conditions.



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  		FIG. 5. Schematic comparison of TRH mRNA expression in the PVN of TRß–/– and TRß2–/– mice under different thyroid states. Data are presented as percentages of TRH mRNA expression in the PVN, relative to 100% expression (hatched line) in euthyroid wild-type control mice. TRß2–/– mice [hatched columns, adapted from Abel et al. (25 )] and TRß–/– mice, lacking both TRß1 and TRß2 isoforms (black columns, presented herein) were compared. In both TRß2–/– and TRß–/– mice, TRH regulation by TH is impaired. In TRß2–/– mice, given PTU treatment or no treatment, relative TRH expression is significantly maintained above the 100% expression control. On the contrary, in TRß–/– mice, relative TRH expression remains significantly below the 100% expression control in the presence of PTU treatment or no treatment (first and second pairs of columns). After T3 treatment, TRH levels are significantly above the 100% untreated control level in both TRß2–/– and TRß–/– mice (third pair of columns).

 
Thus, the ISH and the gene transfer data demonstrate that TRß1 plays a role in ligand-independent TRH activation. This T3-independent activation could possibly rely on the binding of unliganded TRß1 on the major negative TRE of TRH-luc(named site 4). Indeed, by mutating site 4, T3-independent transcriptional activation of TRH-luc was previously found to be abolished in vivo by our group (22) and in vitro by others (43).

Taken together, our ISH and functional data suggest that it is unlikely TRß2 alone that accounts for the major role of the TRß isoforms in TRHtranscriptional regulation.

Coming to the role of TR{alpha}1, previous functional data from our group indicated an active role for TR{alpha}1 in repressing TRH-luc transcription in the absence of T3 (21). This was also suggested by Calza et al. (44), whose findings showed an increased expression of TRH mRNA in untreated mice devoid of all known TRs. Gene transfer studies on hypothyroid TR{alpha}o/o pups were unfeasible, due to the paucity of newborn mice. Thus, to analyze further whether any role could be assigned to endogenous TR{alpha}1, TRH mRNA expression was quantified in the PVN of mutant mice expressing no TR{alpha} isoforms (TR{alpha}o/o), with experimentally modified thyroid status. Indeed, this approach also allows us to follow the effects of TRß isoforms alone on TRHregulation.

A first key observation was that, in hypothyroid TR{alpha}o/o mice, TRH expression is significantly greater than in euthyroid TR{alpha}o/o mice and in hypothyroid wild-type controls. A second is that, in hyperthyroid TR{alpha}o/o mice, the opposite effect is seen: TRH transcription is more efficiently repressed in response to T3 in hyperthyroid TR{alpha}o/o mice than in wild-type control (hyperthyroid) animals. Thus, the TR{alpha}o/o mice display hypersensitivity to TH in terms of TRHregulation. This hypersensitivity has also been described for other target genes (such as malic enzyme) in TR{alpha}o/o mice (31), for which the response to TH was exacerbated. The hypersensitivity of TRHregulation to TH in the PVN of TR{alpha}o/o mutants could rely on different, nonexclusive, events such as overexpression of hypothalamic TRß isoforms in TR{alpha}o/o mutants, as well as the removal of the possible dominant negative effect of TR{alpha}2, and/or the possible repressive effect of TR{alpha}1.

Another gene negatively regulated by T3, via TRs, is the pituitary gene coding for the TSHß subunit. Its expression has recently been studied at the mRNA level in TRß–/– mice. Unlike our observations on the TRHgene, there is no impairment of activation in the absence of TRß1 and TRß2 isoforms (45). Furthermore, again unlike TRH, there is still a T3-dependent repression of TSHßin TRß –/– mice (46). Recently, this remaining T3-dependent repression has been shown to be impaired by pituitary-restricted expression of a dominant negative TRß transgene in TRß –/– mice (29). These results suggest that the TR{alpha}1 isoform participates in the T3-dependent repression of TSHß, at least in the absence of TRß isoforms. We can thus conclude that the T3-dependent regulation of the two key genes of the hypothalamo-pituitary axis are governed by different molecular mechanisms. These differences could rely on tissue-specific factors, such as the relative levels of synthesis of TR isoforms or comodulators, and could also rely on different response elements in the promoters of these genes. Thus, TRHrepresents a distinct model for negative regulation by T3, with a differential role for TRß1 and TRß2 isoforms.


   Acknowledgments
 
We thank Laurent Sachs, Marie-Stéphanie Clerget-Froidevaux and Olivier Chassande for helpful discussions and comments on the manuscript. We thank Jean-Paul Chaumeil, Gérard Rancillac, and their co-workers for animal care.


   Footnotes
 
This work was supported by the Association pour la Recherche Contre le Cancer and European Grant QL 93 CT 2000 00 844. S.M.D. was also a fellow of the Ministère de la Recherche. We thank Ligue Nationale Contre le Cancer for providing grants to S.M.D. and H.G.

Abbreviations: b.w., Body weight; ISH, in situ hybridization; PEI, polyethylenimine; PFA, perifornical area; PTU, 6-n-propyl-2-thiouracil; PVN, paraventricular nucleus of the hypothalamus; TH, thyroid hormone; TR, TH receptor; TRE, TH response element.

Received September 11, 2003.

Accepted for publication January 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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