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cgSpringer Molecular and Cellular Biochemistry 278: 93–100, 2005. 2005 Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain Caterina Constantinou, Marigoula Margarity and Theony Valcana Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras, Greece Received 16 February 2005; accepted 4 May 2005 Abstract The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA+ RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T3 -binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T4 intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005) Key words: adult rat brain, hypothyroidism, methimazole, thyroid hormone receptor isoforms (TR) Introduction Thyroid hormones exert their physiological role mainly through binding to specific nuclear receptors, which are encoded by two different protooncongenes, c-erbAα and c-erbAβ [1]. Each gene has several alternative mRNA splicing products including the predominant isoforms of thyroid hormone receptors, TRα1, TRα2, TRβ1 and TRβ2 [2–6]. TRα1, TRβ1 and TRβ2 bind T3 with high affinity and also bind to thyroid hormone response elements (TREs) on chromatin, regulating thus transcriptional processes in several target tissues, including adult rat brain [7]. Binding of unliganded TRs on positively regulated TREs inhibits the T3 dependent gene expression [8–11]. The above T3 -dependent gene repression is reversed upon binding of T3 on its specific nuclear receptors [12–15]. TRα2 variant, an alternate splice product of the TRα1, cannot bind T3 because it lacks a big part of its ligand binding domain [4, 6, 16]. It has been suggested that this structural differentiation is responsible for its inability to up-regulate T3 -dependent gene expression. In addition, the TRα2 variant, due to its truncated ligand binding domain has been proposed to be inefficient suppressor of target gene expression despite its endogenous and resident unliganded conformation [17, 18]. Address for offprints: M. Margarity, Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras, Greece (E-mail: [email protected]) 94 Quantitative differences in the expression pattern of TR isoforms have been observed in several T3 target tissues in rat, mice and human in both adult and developmental stages [19–22]; it is noticeable that the predominant TR isoform in adult rat brain is the non-T3 -binding variant of TRs (TRα2) [20, 23]. Furthermore, in situ hybridization studies showed that TRα1 and TRα2 were widely distributed in adult rat brain in a similar, almost identical, pattern; their highest levels were observed in the olfactory lobe, hippocampus and granular layer of cerebellar cortex. Expression of TRβ isoforms was located mainly in the paraventricular nuclei of hypothalamus, anterior pituitary and it was a little less evident in cerebral neocortex and hippocampus [24]. The physiological relevance of the differential quantitative distribution of TR isoforms in each brain region is still unknown. Recent knockout and knock-in studies in mice and the use of synthetic TR agonists revealed common as well as divergent actions of the TR isoforms, indicating that the relative expression of each TR isoform in each target tissue may regulate the specific response of these tissues to T3 [25–29]. Knockout studies also showed that hypothyroidism causes a more acrimonious phenotype in mice than the absence of one or all TR isoforms [26, 30–32], suggesting that there is also different physiological relevance for T3 and its nuclear receptors. Recent studies also showed that the relative expression of TRs in adult rat cerebral hemispheres is affected by altered neural activity in an isoform-specific manner, indicating thus that each TR isoform contributes to different physiological response of adult rat brain to TH [33, 34]. Furthermore, these studies showed that the intacellular thyroidal status was important in determining the alterations in the expression pattern of TRs under altered neural activity. Cerebral hemispheres are under tight control of their intracellular levels of TH due to the action of specific deiodinases (5 DII and 5DII) [35] and are also characterized by a large number of nuclear T3 receptors that increases under hypothyroidism [36]. The purpose of this study was to elucidate whether in vivo depletion of TH affects the relative expression of all TR isoforms expressed in adult rat cerebral tissues and consequently to delineate if there is any physiological role of the percent contribution of each isoform upon the whole of TRs. The cerebral tissues used for the present study were the cerebral cortex and hippocampus. Furthermore, for comparative reasons the relative expression of TRs under hypothyroidism was also determined in cerebellum, a less abundant in nuclear T3 receptors brain tissue. To address these questions, a hypothyroid adult rat model that confers effective depletion of serum (peripheral) TH and TH depletion at the synaptosomal (local) level of adult rat cerebral tissues was used. Materials and methods Animals and treatment Adult Wistar rats (50 days) of both sexes were bred in our laboratory, housed four per cage and given laboratory chow and water ad libitum. Animals were treated according to the standards of the international statutes on animal handling (86/609/EEC) and exposed to regular light – dark cycle (light period: 7 a.m. to 7 p.m.; dark period: 7 p.m. to 7 a.m.; at 22 ± 1◦ C) for at least 1–2 weeks prior to methimazole (MMI) treatment and until sacrifice. The animals were rendered hypothyroid by 4.5 weeks of treatment with 0.025% methimozole in their drinking water; the control group was given water ad libitum. A second group of hypothyroid animals received T4 [0.4 mg T4 /kg body weight (BW)] with three different intraperitoneal injections 24, 12, and 4 hours to each hypothyroid animal before sacrifice. Body weights were recorded daily inasmuch as growth inhibition represents a measure of hypothyroidism. After sacrifice, brains were rapidly removed in a sterile cooled glass plate and divided into the following regions: cerebral cortex, hippocampus and cerebellum. Each brain region was rapidly weighed and frozen in liquid N2 until assayed by Northern analysis within 2 weeks. The effectiveness of MMI treatment and administration of T4 , were confirmed by the determination of the serum triiodothyronine (T3 ) and thyroxin (T4 ) with radioimmunoassay in both male and female animals, since it is reported that after the 21th day of MMI treatment (0.03% in the drinking water) of young (2–3 months old) Wistar rats, serum TH levels are similarly decreased in both genders [37]. The specific radioimmunoassay kits (T3 -RIA and T4 -RIA) were supplied from the Hellenic Center of Natural Research, “Demokritos”). Isolation of cerebral synaptosomes and determination of the thyroid hormone synaptosomal levels The preparation of synaptosomes was performed according to the method of Sarkar and Ray [38] with slight modifications [33]. Briefly, the cerebral hemispheres (∼ =800 mg) were homogenized (10%, w/v) in ice-cold 0.32 M sucrose (pH 7.4). The homogenate was then centrifuged at 1000×g for 10 min, the supernatant was layered slowly on top of a sucrose gradient composed of 8 ml of 1.20 M and 8 ml of 0.32 M sucrose (4 ◦ C, pH 7.4) and was centrifuged at 34,000 × g for 50 min. The crude synaptosomal fraction was banded between 0.32 and 1.20 M sucrose layers and was carefully removed by suction at 4 ◦ C. The sucrose concentration was adjusted to 0.32 M by slowly adding ice-cold bidistilled water (∼ =1:1.8). Then the samples were layered slowly on top of a sucrose gradient 95 composed of 8 ml of 0.85 M and 8 ml of 0.32 M sucrose (4 ◦ C, pH 7.4) and centrifuged at 34,000× g for 30 min. The bottom pellet thus obtained was the synaptosomal fraction, which was further purified by washing once with 5 ml 0.32 M sucrose at 4 ◦ C and repelleted at 20,000× g for 20 min to finally get pure synaptosomes. The synaptosomes were then ruptured hypo-osmotically in 1.5 ml of 5.5 mM imidazole–HCl buffer (pH 7.4, 4 ◦ C). The ruptured synaptosomal suspensions were used immediately for T4 and T3 radioimmunoassay utilizing specific kits (T3 -RIA and T4 -RIA) from the Hellenic Center of Natural Research, Demokritos. Prior to analysis, in each synaptosomal suspension sample (100 µl) 10 µl of 0.1 N NaOH was added in order to obtain an appropriate alkaline environment for the dissolution of thyroid hormones from the membrane fraction of synaptosomes. All samples were analyzed in triplicate. Protein concentration was evaluated by the method of Bradford [39], using bovine serum albumin as standard. PolyA+ RNA extraction PolyA+ RNA was extracted from 1 g of each brain region and experimental condition using the Poly(A)PureTM kit (Ambion), which is based on the guanidine thiocyanate method. The final RNA pellets were dissolved in RNase free water and quantified in duplicate by determining the O.D. at 260 nm. In each experiment three different polyA+ RNA preparations from each brain region and experimental condition were analyzed by Northern blot hybridization twice for each TR isoform mRNA. Northern blot hybridization The relative expression of each TR isoform was determined by Northern blot hybridization according to the method of Sambrook et al. [40]. Briefly, aliquots of polyA+ RNA, appropriate for each TR isoform, were subjected to electrophoresis in a 1% agarose/formaldehyde gel (20 mM 3-[n-morpholino]2-hydroxy-propanesulfonic acid (MOPS), 1 mM ethylene diamine tetraacetic acid (EDTA), 5 mM sodium acetate, pH 7.0, and 2.2 M formaldehyde). The mRNA was transferred overnight to positively charged nylon membranes (NEN) and subsequently cross-linked by baking the membranes at 80 ◦ C for 2 h. The blots were prehybridized at 68 ◦ C in UltraHyb hybridization solution (Ambion) for 2 h. Hybridization was done overnight at 62 ◦ C for TRα1 isoform, 68 ◦ C for TRα2 isoform and 65 ◦ C for TRβ1 and TRβ2 isoforms with 3×106 cpm/ml of random labeled probes arising by digestion of the TRα1, TRα2 and TRβ2 full-length cDNAs with restriction enzymes (α1 specific probe: EcoRI and XbaI in α1 cDNA, α2 specific probe: EcoRI and XhoI in α2 cDNA, β1/β2 common probe and β2 specific probe: XbaI in β2 cDNA). Blots were washed twice in 2 × standard saline citrate buffer–SSC (30 mM sodium citrate, 300 mM sodium chloride, pH 7.0)/0.1% sodium dodecyl sulphate (SDS) at 62 ◦ C for 20 min, twice in 0.1 × SSC/0.1% SDS at 65 ◦ C for 10 min and finally in 0.1 × SSC at room temperature for 1–2 min. The washed blots were exposed immediately to the Phosphor imager cassette (Molecular Dynamics) for 24 h and quantified by computer-assisted densitometry. The size of each thyroid hormone receptor isoform mRNA was determined by comparison with RNA molecular weight markers (Promega). PolyA+ RNA recovery was determined by rehybridization of membranes with the glyceraldhehyde-3phosphate-dehydrogenase (GAPDH) probe and all values were normalized to the GAPDH signal. Statistical analysis Results are expressed as the mean ± S.E. and analyzed statistically by performing one-way completely randomized analysis of variance (ANOVA) with a Bonferroni correction for multiple comparisons and the p < 0.05 as the significance cutoff point. Results Effects of adult hypothyroidism on the serum T3 and T4 levels The progress of the methimazole-induced hypothyroidism and T4 replacement in the hypothyroid rats were evaluated by the determination of the serum T3 and T4 levels with specific radioimmunoassays. As shown in Table 1, 4.5 weeks of methimazole treatment decreased both T4 and T3 levels in blood circulation (74.9 and 81.9%, respectively). T4 replacement in the hypothyroid rats reversed the above effects of methimazole treatment to almost euthyroid levels. Effects of adult hypothyroidism on the TH synaptosomal levels from cerebral hemispheres In agreement with our previous results [33, 34] and with data from the literature [41], the T4 synaptosomal levels in cerebral hemispheres remained undetectable in all control and experimental animals. However, a decrease (≈20%) in the T3 synaptosomal levels was observed in cerebral hemispheres of hypothyroid rats (Table 2), indicating a progressive induction of hypothyroidism at the cellular level in adult rat brain. 96 Table 1. Effects of hypothyroidism on the serum T4 and T3 levels T3 (ng/ml) Effects of adult hypothyroidism on the relative expression of TRα1 in cerebral cortex, hippocampus and cerebellum T4 (ng/ml) Euthyroid n = 70 0.882 ± 0.1 Hypothyroid (methimazole 4.5 weeks) n = 40 0.16 ± 0.09 (↓ 81.9%) 33 ± 4 ∗ ∗ 8.33 ± 1 (↓ 74.9%) Hypothyroid + T4 (methimazole +T4 , 24, 12 and 4 h before sacrifice) n = 40 ∗ 0.755 ± 0.13 (↓ 14.4%) ∗ 29.2 ± 2 (↓ 11.5%) TRα1 isoform was expressed in all brain regions we examined (Fig. 1A). In euthyroid animals TRα1 isoform was expressed in similar levels in cerebral cortex and hippocampus, while statistically lower levels (14%, p < 0.05) were recorded in cerebellum (Fig. 1). The levels of TRα1 mRNA in hypothyroidism were increased in all brain regions examined and particularly in hippocampus (46.6%) and cerebral cortex (38%). A small, statistically not significant ( p > 0.05) increase (12%) of TRα1 mRNA levels in cerebellum under hypothyroidism was also observed. It was noticed that all the Note. Numbers represent means ± S.E. of n preparations (one animal per preparation) assayed in triplicate. ∗ Statistically different from euthyroid ( p < 0.05, ANOVA with a Bonferroni correction for multiple comparisons). Table 2. Effects of hypothyroidism on the cerebral hemisphere synaptosomal T3 levels T3 (ng/mg protein) Euthyroid n = 14 0.478 ± 0.006 Hypothyroid (methimazole 4.5 weeks) n = 19 0.376 ± 0.03 ∗ Hypothyroid + T4 (methimazole + T4 ) n = 15 0.432 ± 0.02 Note. Numbers represent means ± S.E. of n preparations (one animal per preparation) assayed in triplicate. ∗ Statistically different from euthyroid ( p < 0.05, ANOVA with a Bonferroni correction for multiple comparisons). Effects of adult hypothyroidism on the relative expression of TRβ1 in cerebral cortex, hippocampus and cerebellum Northern blot hybridization was used in order to evaluate the relative expression of TRβ1 mRNA in various brain regions under hypothyroidism. TRβ1’s relative expression is stronger in the cerebral cortex (4560 ± 182 AU/µg polyA+ RNA) than in hippocampus (2081 ± 177 AU/µg polyA+ RNA) of euthyroid rats, whereas it is not detectable in the cerebellum of both eu- and hypothyroid animals. The later observation is in agreement with previous studies suggesting that both TRβ1 and TRβ2 isoforms are not detectable in cerebellum by Northern analysis [20]. Hypothyroidism did not alter TRβ1’s relative expression in cerebral cortex and hippocampus of hypothyroid animals (data not shown). TRβ2 isoform was not detectable by Northern analysis in any of the brain regions we examined, in both eu- and hypothyroid animals, which is in agreement with previous studies [5, 20]. Fig. 1. (A) (Top) Northern blot analysis of TRα1 isoform in various brain regions of adult hypothyroid rats. Eight micrograms of polyA+ RNA preparations from cerebral hemispheres of control, hypothyroid (methimazoletreated) and hypothyroid animals which received T4 , were applied in 1% agarose/formaldehyde gel. Lanes 1–3: cerebral cortex (1, hypothyroid; 2, control; 3, hypothyroid + T4 ); Lanes 4–6: hippocampus (4, hypothyroid; 5, control; 6, hypothyroid + T4 ); Lanes 7–9: cerebellum (7, hypothyroid; 8, control; 9, hypothyroid + T4 ). (Bottom) PolyA+ RNA recovery was determined by rehybridization of the membrane with the GAPDH probe and all values were normalized to the GAPDH signal. (B) Effects of hypothyroidism on the relative expression of the TRα1 mRNA levels in various brain regions of adult rats. Northern blots from five separate experiments were quantified as described under Materials and methods. All data are expressed as arbitrary units (AU) per µg polyA+ RNA and all values represent means ± S.E. of five different experiments. (∗ ) Statistically different from control ( p < 0.05, ANOVA with Bonferroni correction for multiple comparisons). 97 above mentioned changes returned to euthyroid levels following intraperitoneal T4 administration to the hypothyroid animals (Fig. 1). Effects of adult hypothyroidism on the relative expression of TRα2 in cerebral cortex, hippocampus and cerebellum Figure 2A shows representative results of Northern blot hybridization for TRα2 mRNA in cerebral cortex, hippocampus and cerebellum of euthyroid, hypothyroid and hypothyroid animals which received T4 . TRα2 variant (non-T3 -binding) was expressed in all brain regions examined; however, the TRα2 mRNA was more evident in hippocampus than in cerebral cortex (two times) and cerebellum (four times) of euthyroid animals (Fig. 2). In hypothyroidism, the TRα2 mRNA levels were decreased in all brain regions examined. However, the percent decrease of TRα2 mRNA in hypothyroid animals is similar in cerebral cortex and hippocampus (26.4 and 27.3%, respectively) whereas a smaller, statistically significant ( p < 0.05) decrease (10.9%) of the TRα2 relative expression was observed in cerebellum. All the above observed changes returned to normal following T4 replacement of hypothyroid animals. The observed decrease in TRα2 mRNA levels in cerebral cortex and hippocampus was inversely proportional to the observed increase of the number of the in vitro T3 binding sites on cerebral nuclei of hypothyroid rats [36, 42, 43]. Discussion Fig. 2. (A) (Top) Northern blot analysis of TRα2 isoform in various brain regions of adult hypothyroid rats. three micrograms of polyA+ RNA preparations from cerebral hemispheres of control, hypothyroid (methimazoletreated) and hypothyroid animals which received T4 , were applied in 1% agarose/formaldehyde gel. Lanes 1–3: cerebral cortex (1, hypothyroid; 2, control; 3, hypothyroid + T4 ); Lanes 4–6: hippocampus (4, hypothyroid; 5, control; 6, hypothyroid + T4 ); Lanes 7–9: cerebellum (7, hypothyroid; 8, control; 9, hypothyroid + T4 ). (Bottom) PolyA+ RNA recovery was determined by rehybridization of the membrane with the GAPDH probe and all values were normalized to the GAPDH signal. (B) Effects of hypothyroidism on the relative expression of the TRα2 mRNA levels in various brain regions of adult rats. Northern blots from five separate experiments were quantified as described under Materials and methods. All data are expressed as arbitrary units (AU) per µg polyA+ RNA and all values represent means ± S.E. of five different experiments. (∗ ) Statistically different from control ( p < 0.05, ANOVA with Bonferroni correction for multiple comparisons). The main object of this study was to delineate whether in vivo depletion of TH affects the TR expression pattern in several adult rat brain regions. Hypothyroidism was evaluated by the determination of the serum TH levels and by the determination of the intracellular cerebral tissue TH availability. Our data revealed that MMI treatment for 4.5 weeks decreases the plasma T3 and T4 levels by 82 and 75%, respectively. This alteration should not be attributed to direct pharmacological effect of MMI on the serum TH levels, since T4 replacement in the hypothyroid rats reversed the above effects of MMI treatment to almost euthyroid levels. The intracellular thyroid status in adult cerebral tissue was evaluated by the determination of the endogenous synaptosomal TH levels; T3 , the active form of TH, is concentrated in the nerve ending fractions of adult rat brain [44], deriving presumably from the local deiodination of T4 [44, 45]. Our results showed that MMI treatment induced hypothyroidism also at the cellular level in adult rat cerebral hemispheres but in a lesser extent than in plasma. The milder hypothyroidism in cerebral tissue could be attributed to the induction of the local deiodination of T4 to T3 under hypothyroidism [35]. Our study revealed that TRα1 isoform (T3 -binding isoform) is expressed in similar levels in cerebral cortex and hippocampus, whereas it is less evident in cerebellum of euthyroid rats. TRβ1 (also T3 -binding isoform) follows an inverse expression pattern in comparison to that of TRα1, being more evident in cerebral cortex than in hippocampus and undetectable in cerebellar tissue. The only T3 -binding isoform detected in cerebellum by Northern analysis in both eu- and hypothyroid adult rats was the TRα1, explaining thus the relatively lower T3 -binding capacity (Bmax ) observed in this brain region compared to the T3 Bmax in cerebral cortex and pituitary [46]. The above observations are in agreement 98 with the in situ hybridization findings of Bradley et al. [24]. A high abundance of the TRα2 (non-T3 -binding isoform) in all brain regions we examined was demonstrated, which is in agreement with previous studies showing that this TR variant is the predominant isoform of TRs in adult brain [20, 23]. However, quantitative region-specific differences in TRα2 relative expression were also observed in euthyroid animals, indicating that the TRα1/TRα2 mRNA ratio is differentially regulated, in a region specific manner in euthyroid adult rat brain; the lower ratio was found in hippocampus and the highest in cerebellum. In hypothyroidism the changes of the TRα1 and TRα2 relative expression are inversed in all brain regions examined. That is, while there is an induction in TRα1 mRNA levels, the TRα2 mRNA levels are decreased, despite the fact that both of these TR isoforms are coded on the same gene (cerbAα) and generated by alternative splicing procedure [2, 4]. This observation is in agreement with the literature, which indicates that these alternative splicing products (TRα1 and TRα2) of the c-erbAα gene are related inversely [47]. The molecular mechanism, which regulates the expression and/or the alternative splicing of the c-erbAα gene, is still unknown. It has been hypothesized that the Rev-ErbAα mRNA – an endogenous antisense RNA to the TRα2 mRNA – controls the TRα2 mRNA levels through base-pairing interactions [47, 48]. However, this hypothesis remains unverified in vivo. Additionally, it has been shown that T3 treatment of euthyroid animals decreases the total expression of the c-erbAα gene without affecting the TRα1/TRα2 mRNA ratio in many T3 responding tissues with the exception of brain [20, 23, 49]. Our results show that the absence of thyroid hormones affects differentially the TRα1 and TRα2 mRNA levels in cerebral cortex, hippocampus and cerebellum. Furthermore, quantitative differences in the percent change of each TRα isoform in a region-specific manner under hypothyroidism were also observed. However, recent studies in mice indicate that hypothyroidism (propylthiouracil treatment for 13 days) does not affect the relative expression of TRα1, TRα2 and TRβ1 isoforms in motor and cingulate cortex [22]. Nevertheless, this divergence could be attributed to the two novel, TR isoforms, identified in mice and not in rats, the TRα1 and TRα2 [50]. Adult rat brain, relative to other tissues, contains a high density of specific nuclear T3 -binding receptors, the number of which increases in response to hypothyroidism [36, 43]. Our results show that this increase of the number of the in vitro T3 -binding sites on brain DNA could only be due to the observed increase of the TRα1 mRNA levels in all brain regions, since the expression of TRβ1 levels remained unaffected in cerebral cortex and hippocampus and not detectable in cerebellum of hypothyroid animals. The percent reduction in TRα2 mRNA levels in cerebral cortex and hippocampus of hypothyroid animals was inversely proportional to the previously observed increase of the in vitro T3 -binding sites in cerebral nuclei [36, 43]. This observation is in agreement with our previous studies showing that the relative expression of TRα2 variant is always inversely proportional to the observed alteration in T3 Bmax in cerebral hemispheres under perturbed neural activity [33, 34]. These observations indicate that the regulation of the non-T3 -binding variant of TRs could have a significant role in the determination of the maximal number of nuclear T3 -binding sites in adult hypothyroid brain. In support to this hypothesis there are previous reports, showing that TRα2 inhibits the T3 -dependent transactivation of TRβ and TRα1 – a phenomenon termed the dominant negative effect of TRα2 [18, 51, 52] – due to competitive substitution with the T3 -binding TR isoforms on the same TREs in chromatin [53]; its negative efficacy however, is less potent than that of the unliganded T3 -binding isoforms [17, 18]. The reduction of the TRα2 mRNA levels in brain tissue of hypothyroid animals could consequently, lead to a release of DNA-binding sites available for the T3 -binding TR isoforms – increasing thus the maximal number of the in vitro T3 -binding sites in adult rat brain DNA. These DNA-binding sites must be regarded as direct repeat (DR) TREs that contain two additional nucleotides [T(A/G)] before the down stream hexamer AGGT(C/A)A or DR TREs that contain the two additional nucleotides mentioned above in both the hexamers participating in the TRE; it has been shown that only these two species of TREs can bind the TRα2 variant with high affinity and stability, allowing thus the TRα2 isoform to regulate the T3 -target genes in a T3 -independent manner [17, 53]. This potential function should be regarded in a selective manner dependent upon the restrictions of TRα2 ability to bind on particular types of TREs; T3 -responsive genes that have the optimal DR TRE for TRα2 binding could be transcriptionally regulated to stable levels (T3 independently) by the TRα2 variant, whereas other T3 -target genes would escape TRα2 T3 -independent regulation. However, this manner of transcriptional regulation may provide a molecular switch that is controlled by the relative amounts of TRα2 and the T3 -binding isoforms of TRs; the dominant negative effect of TRα2 is related to its increased expression [6, 54]. The final resolution, however, of the physiological relevance of the TRα2 variant will depend upon the determination whether the above described alterations in the TR expression pattern in several brain regions under hypothyroidism reflect into changes in the respective proteins in the nucleus. In addition, the identification of the specific T3 target genes, which are under the potential transcriptional control of TRα2, would provide direct in vivo evidence for its functional contribution to the TH molecular mechanism of action. 99 Acknowledgments The authors gratefully acknowledge Prof. W.W. 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