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Acute LiCl-treatment affects the cytoplasmic T4 availability and the expression pattern of thyroid hormone receptors in adult rat cerebral hemispheres
Neuroscience Research 51 (2005) 235–241 www.elsevier.com/locate/neures
Acute LiCl-treatment affects the cytoplasmic T4 availability and the expression pattern of thyroid hormone receptors in adult rat cerebral hemispheres Caterina Constantinou, Stamatis Bolaris1, Theony Valcana, Marigoula Margarity* Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras 265 00, Greece Received 6 October 2004; accepted 22 November 2004 Available online 7 January 2005
Abstract We have previously reported that short-term LiCl-treatment affects the kinetic characteristics of thyroid hormone binding in adult rat brain (Bolaris, S., Margarity, M., Valcana, T., 1995. Effects of LiCl on triiodothyronine (T3) binding to nuclei from rat cerebral hemispheres. Biol. Psychiatry 37, 106–111); however, the mechanism underlying the above effects of LiCl administration is yet to be determined. In this study, the effects of lithium within one day after its administration (5 mmol/kg BW) on the relative expression of thyroid hormone receptor isoforms and on the cytoplasmic and synaptosomal thyroid hormone availability in adult rat cerebral hemispheres were examined. Although short-term LiCl-treatment did not affect the levels of triiodothyronine either in the synaptosomal or in the cytoplasmic fraction 24 h after LiCl administration, the cytoplasmic availability of thyroxin was lower. In addition, 24 h after the administration of lithium the mRNA levels of the TRa1 isoform (T3 binding) increased while the relative expression of the TRa2 variant (non-T3 binding) was decreased. Notably, the decrease of the TRa2 mRNA levels was also observed 4 h after LiCl administration. The expression levels of the TRb1 isoform were unaffected in any interval examined. The present study suggests that short-term lithium treatment regulates the relative expression of TRs in an isoform-specific manner and affects the cytoplasmic availability of thyroxin in adult rat brain. # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Lithium (LiCl); Adult rat brain; Thyroid hormone receptors (TR); Triiodothyronine (T3); Thyroxin (T4); Thyroxine availability
1. Introduction Bipolar disorder (manic-depressive illness) is a common psychiatric condition characterized by cycling episodes of mania and severe depression; however, the pathophysiological mechanism(s) underlying bipolar disorder is not completely understood (Goodwin and Jamison, 1990) although molecular genetic studies of this disorder have been proceeded (Kato, 2001). The efficacy of lithium in the treatment of bipolar disorder is well established (Cade, 1949; Frost and Messiha, 1983) and although many cellular effects of lithium have been described, the basis of its * Corresponding author. Tel.: +30 2610 997430; fax: +30 2610 969273. E-mail address: [email protected] (M. Margarity). 1 Present address: Department of Human Reproduction, ‘‘Helena Venizelou’’ Hospital, 2 Venizelou Sqr. 115 21 Athens, Greece.
therapeutic action is unknown (Lenox et al., 1998). Numerous studies have put forward several theories regarding the mechanism(s) underling LiCl therapeutic action, ranging from ion transport to gene expression. Early research focused on LiCl-dependent modulation of presynaptic events and on its effects in postsynaptic intracellular phenomena, involving mainly the cAMP and the phosphoinositide signaling transduction pathways (Brunello, 2004). Recently, it has been shown that lithium treatment confers changes in gene expression (Ausgari et al., 1998) that may account for its therapeutic efficacy in bipolar disorder. A strong correlation between the long-term LiCltreatment and the thyroid hormones (TH) has been well established; chronic lithium administration affects the production and release of TH from the thyroid gland (Burrow et al., 1971; Spaulding et al., 1972; Mori et al.,
0168-0102/$ – see front matter # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2004.11.005
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1989) and the peripheral TH metabolism, including brain tissue (Bagchi et al., 1978; St. Germain, 1987; Baumgartner et al., 1994). In addition, a well-documented theory regarding the therapeutic action of lithium in bipolar disorder has already been proposed (Bolaris et al., 1995). Triiodothyronine (T3), the active form of TH, exerts its physiological role, at the cellular level, mainly through binding to specific nuclear receptors; the TRa1, TRa2, TRb1 and TRb2 isoforms (Thompson et al., 1987; Koenig et al., 1988; Mitsuhashi et al., 1988; Hodin et al., 1989). The TRa1, TRb1 and TRb2 bind T3 with high affinity and also bind to thyroid hormone response elements (TREs) on chromatin, regulating transcriptional processes in several target tissues, including adult rat brain. Binding of unliganded TRa1, TRb1 and TRb2 on TREs represses the basal transcription of the positively regulated T3-target genes; the above described repression is reversed upon binding of T3 to its specific nuclear receptors (Brent et al., 1991; Zhang et al., 1991; Baniahmad et al., 1992; Yen et al., 1993; Li et al., 1999; Wu and Koenig, 2000; Ito and Roeder, 2001; Yen, 2001). Additionally, several negatively regulated T3-target genes have been already identified (Katz and Koenig, 1994; Wu and Koenig, 2000); the mechanism through which the unliganded TRs activate basal transcription of these genes remains unknown, although the involvement of corepressors has been proposed (Tagami et al., 1997, 1999). TRa2, an alternate splice product of the TRa1, cannot bind T3 because it lacks an intact ninth heptad hydrophobic repeat in its ligand binding domain and acquires a unique carboxyl-terminal region (Mitsuhashi et al., 1988; Lazar et al., 1988; Koenig et al., 1989; Katz et al., 1992). The physiological role of this non-T3 binding isoform of TRs remains unknown. The regulation of gene expression has been proposed to be one of the potential mechanisms by which lithium exerts its mood-stabilizing effects (Brunello, 2004). Lithium is capable of regulating the expression of a number of genes in rat brain and cultured-cell models (Sivam et al., 1988, 1989; Colin et al., 1991; Wang and Young, 1996; Miller and Mathe, 1997; Feinstein, 1998; Hua et al., 2000); the c-fos and TR genes, encoding for transcription factors, are among the lithium-regulated genes (Miller and Mathe, 1997; Ausgari et al., 1998; Hahn et al., 1999). However, different effects of lithium on c-fos expression in acute, short-term and long-term lithium treatment have been reported (Miller and Mathe, 1997; Ausgari et al., 1998; Moorman and Leslie, 1998); consequently lithium could affect differentially the down-stream of c-fos gene expression, promoting the cellular adaptation in critical neuronal circuits that lead to mood stabilization. As mentioned above, long-term administration of lithium affects the expression pattern of the thyroid hormone receptors (TR) in several brain areas of adult rats (Hahn et al., 1999). In addition, our previous study has shown an increase of the total nuclear T3 binding after long-term administration of LiCl in adult rat cerebral tissue (Bolaris
et al., 1995). However, it is important to delineate whether the above described effects of lithium on the TH mechanism of action can be also observed following acute administration of this mood stabilizer; this will help to elucidate whether the T3-dependent gene expression is acute responded to lithium. We have previously reported that short-term (24 h) LiCl-treatment increased the in vitro total T3 binding in adult rat cerebral nuclei; an effect that was attributed to an increase of the maximal number of the nuclear T3 binding sites (Bmax), whereas the binding affinity was unaffected (Bolaris et al., 1995). The object of this study was to investigate whether the changes in the nuclear T3 Bmax in adult rat cerebral tissue 24 h after LiCl administration correlate with changes in the expression pattern of the TR isoforms. The cytoplasmic conversion of thyroxin (T4) to T3 and the synaptosomal availability of TH in adult rat cerebral hemispheres were also examined, in order to evaluate the thyroidal status of this tissue after shortterm treatment with lithium.
2. Materials and methods 2.1. Animals and treatments Adult Wistar rats (40 days) of both sexes were bred in our laboratory, were housed 4 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 am to 7 pm; dark period: 7 pm to 7 am; at 22 1 8C) for at least 1–2 weeks prior to LiCl-treatment and until sacrifice. Groups of experimental animals received an intraperitoneal injection of LiCl [5 mmol/kg of body weight (BW)] at 4 and 24 h prior to sacrifice. In all groups, the control animals were injected with an equal volume of 0.9% NaCl. For the study of the in vivo cytoplasmic thyroxin deiodination 24 h LiCl-treated and control animals were injected intraperitoneally with trace 125I-T4 (5 ng/100 g BW; 4400 Ci/mmol, NEN) 4 h prior to sacrifice. After sacrifice, brains were rapidly removed in a sterile cooled glass plate and the cerebral hemispheres were isolated and weighed. For the determination of synaptosomal and cytoplasmic thyroid hormone levels fresh cerebral hemisphere tissue was used. For the mRNA isolation, tissue samples were frozen in liquid N2 until assayed by Northern analysis within 2 weeks. Plasma T3 and T4 levels were determined with specific radioimmunoassay kits (T3-RIA and T4-RIA) from the Hellenic Center of Natural Research, ‘‘Demokritos’’. 2.2. 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 (1992) with slight modifications. Briefly, the cerebral hemispheres
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(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 8C, pH 7.4) and was centrifuged at 34,000 g for 50 min. The crude synaptosomal fraction was banded between the 0.32 and 1.20 M sucrose layers and was carefully removed by suction at 4 8C. 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 composed of 8 ml of 0.85 M and 8 ml of 0.32 M sucrose (4 8C, 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 8C 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 (4 8C, pH 7.4). 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 ml) 10 ml 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 (1976), using bovine serum albumin as standard. 2.3. Identification of cytoplasmic iodothyronines For the study of the in vivo identification of cytoplasmic iodocompounds, 24 h LiCl-treated and control animals were injected intraperitoneally with trace 125I-T4 (5 ng/100 g BW; 4400 Ci/mmol, NEN) 4 h prior to sacrifice. Cerebral hemispheres from two animals were dissected (4 8C), weighed and homogenized in 0.32 M cold sucrose solution (20% w/v, glass-teflon homogenizer, 1000 rpm, 4 8C). After sampling in duplicate for the total radioactivity, the homogenate was centrifuged at 1000 g for 10 min at 4 8C. The supernatant, after addition of dithiothreitol to a final concentration of 5 mM, was immediately centrifuged for 90 min at 100,000 g at 4 8C. The supernatant of this centrifugation (cytoplasmic fraction) was sampled in duplicate and each sample was used for the determination of protein and the distribution of 125I-T3 and 125I-T4 among total radioactivity. For this purpose, the cytoplasmic fraction was treated with 95% ethanol until 85% or more of the radioactivity contained was extracted. These extracts were evaporated to dryness under nitrogen in a 37 8C water bath in the dark. The residue of these samples was dissolved in a mixture of methanol/concentrated NH3 (3:1, v/v). The iodocompounds of these extracts were identified and quantified by thin-layer chromatography (TLC) as pre-
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viously described by Heider and Bronk (1956). The protein content of the cytoplasmic fraction was determined by the method of Lowry et al. (1951). 2.4. Northern blot hybridization The relative expression of each thyroid hormone receptor isoform was determined by Northern blot hybridization. PolyA+RNA was extracted from 1 g of cerebral hemispheres from LiCl-treated and control animals utilizing the Poly(A)PureTM kit (Ambion). In each experiment three different polyA+RNA preparations from LiCl-treated and control animals were analyzed by Northern blot hybridization twice in duplicate for each TR isoform mRNA as described previously by Sambrook et al. (1989). Briefly, after electrophoresis of polyA+RNA samples in 1% agarose/ formaldehyde gel, the electrophoretic-separated mRNA was transferred overnight to positively charged nylon membranes (NEN) and subsequently cross-linked by baking the membranes at 80 8C for 2 h. The blots were prehybridized at 68 8C in UltraHyb hybridization solution (Ambion) for 2 h. Hybridization was performed overnight at 62 8C for TRa1 isoform, 68 8C for TRa2 isoform and 65 8C for TRb1 and TRb2 isoforms with 3 106 cpm/ml of random labeled probes arising by digestion of the TRa1, TRa2 and TRb2 fulllength cDNAs, with restriction enzymes (a1 specific probe: EcoRI and XbaI in a1 cDNA, a2 specific probe: EcoRI and XhoI in a2 cDNA, b1/b2 common probe and b2 specific probe: XbaI in b2 cDNA). Blots were washed according to the instructions of NEN for the particular membrane used and then were exposed immediately to the Phosphorimager cassette (Molecular Dynamics) for 24 h and quantified by computer-assisted densitometry. PolyA+RNA recovery was determined by rehybridization of the membranes with the glyceraldhehyde-3-phosphate-dehydrogenase (GAPDH) probe and all values were normalized to the GAPDH signal. 2.5. Statistics Results were expressed as the mean S.E., analyzed statistically by performing one-way completely randomized analysis of variance (ANOVA) with the P < 0.05 as the significance cutoff point. One-way analysis of variance with a Bonferroni correction for multiple comparisons was performed for the effect of short-term LiCl-treatment on the relative expression of all TR isoforms and on T3 synaptosomal levels.
3. Results 3.1. The effects of short-time LiCl-treatment on the thyroid hormone synaptosomal and cytoplasmic levels Administration of LiCl for 4 and 24 h had no significant effect on plasma T4 and T3 levels (T4: 33.4 2 ng/ml; T3:
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0.734 0.1 ng/ml). These findings are in agreement with our previous data (Bolaris et al., 1995). The determination of the iodothyronines (125I-T3 and 125 I-T4), following in vivo 125I-T4 injection in the total cytoplasmic fraction of adult rat cerebral hemispheres is presented in Table 1. These data show that 24 h of LiCltreatment had no effect on the 125I-T3 levels (125I-T3: 53.9 8.4 106 ng/mg protein) resulting from the local deiodination of 125I-T4 [T3(T4)]; however, the levels of 125I-T4 were decreased (47%) in the same intracellular fraction (Control 125I-T4: 108.5 22.5 106 ng/mg protein; LiCl-treated 125I-T4: 57.4 3.1 106 ng/mg protein, P < 0.01). Short-term LiCl-treatment (5 mmol/kg BW, 4 and 24 h prior to sacrifice) had no effect on the synaptosomal levels (0.473 0.05 ng/mg protein) of the active form of thyroid hormones (T3). In agreement with data from the literature (Sarkar and Ray, 1994) the T4 synaptosomal levels remained undetectable in all control and LiCl-treated animals examined in this study. 3.2. The effects of short-time LiCl-treatment on the relative expression of all thyroid hormone receptor isoforms in adult rat cerebral hemispheres Northern blot hybridization was employed in order to study the effect of short-term LiCl-treatment on the relative expression of TRs in cerebral hemispheres from control and LiCl-treated animals (5 mmol/kg BW, 24 and 4 h prior to sacrifice). Our results show that 24 h of LiCl-treatment increased the TRa1 (T3 binding) relative expression (12%) (Fig. 1), while the TRa2 variant’s (non-T3 binding) mRNA levels were decreased (38%) (Fig. 2). A reduction of the TRa2 relative expression was also observed 4 h (16%) after LiCl administration, whereas the TRa1 mRNA levels were unaffected at this interval. In addition, the expression levels of the TRb1 isoform (T3 binding) were not affected in any interval examined within 24 h after LiCl administration. Moreover, in agreement with results of previous studies (Hodin et al., 1989; Strait et al., 1990) the TRb2 isoform could not be detected by Northern analysis in cerebral
Table 1 Effects of 24 h LiCl-treatment on the cytoplasmic T4 deiodination in cerebral hemispheres of adult rats injected with 125I-T4
Control LiCl-treated
125
125
125
I-T4 (106ng/mg protein)
I-T3( I-T4) (106ng/mg protein)
108.5 22.5 57.4 3.1* (# 47%)
53.9 8.4 59.1 13
The LiCl-treated animals received LiCl (5 mmol/kg BW) 24 h prior to sacrifice. Control animals received an equal volume of 0.9% NaCl. All control and LiCl-treated animals received also trace of 125I-T4 (5 ng/100 g BW; spec. activ. 4400 Ci/mmol, NEN) 4 h prior to sacrifice. Numbers represent means S.E. of four preparations (one animal per preparation) assayed in triplicate. * Statistically different from control (P < 0.01, analysis of variance).
Fig. 1. (A) (Top) Northern blot analysis of TRa1 variant from adult rat cerebral hemispheres. 5 mg of polyA+RNA preparations from cerebral hemispheres of control and LiCl-treated (5 mmol/kg BW, 4 and 24 h prior to sacrifice) adult rats were applied in 1% agarose/formaldehyde gel. Lanes 1, 2: control; Lanes 3, 4: LiCl-treated, 4 h; Lanes 5, 6: LiCl-treated, 24 h. (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) Effect of short-term LiCl-treatment on the relative expression of the TRa1 mRNA levels in adult rat cerebral hemispheres. Northern blots from five separate experiments quantified as described under Section 2. All data are expressed as arbitrary units (AU) per mg polyA+RNA and all values represent means S.E. of five different experiments. *Statistically different from control (P < 0.05, analysis of variance).
hemispheres of all control or LiCl-treated animals (data not shown).
4. Discussion The regulation of the T3-dependent gene expression is directly dependent upon the cytoplasmic TH availability and the density of the T3 nuclear receptors. The purpose of this study was the determination of the intracellular thyroidal status of adult rat brain, as well as to examine whether there is a correlation between the observed increase of the number of the nuclear T3 receptors (Bolaris et al., 1995) and the expression pattern of the TR isoforms in adult rat cerebral hemispheres after short-term (within 1 day) LiCl-treatment. Our results show that the cytoplasmic levels of T3(T4) were not affected; however, a reduction (47%) in the
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Fig. 2. (A) (Top) Northern blot analysis of TRa2 variant from adult rat cerebral hemispheres. 5 mg of polyA+RNA preparations from cerebral hemispheres of control and LiCl-treated (5 mmol/kg BW, 4 and 24 h prior to sacrifice) adult rats were applied in 1% agarose/formaldehyde gel. Lanes 1, 2: Control; Lanes 3, 4: LiCl-treated, 4 h; Lanes 5, 6: LiCl-treated, 24 h. (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) Effect of short-term LiCl-treatment on the relative expression of the TRa2 mRNA levels in adult rat cerebral hemispheres. Northern blots from five separate experiments quantified as described under Section 2. All data are expressed as arbitrary units (AU) per mg polyA+RNA and all values represent means S.E. of five different experiments. *Statistically different from control (P < 0.05, analysis of variance).
cytoplasmic levels of T4 was observed in the absence of any alteration in the plasma and synaptosomal TH levels. The above LiCl-induced decrease of cytoplasmic T4 levels could be attributed to a reduction in the T4 intracellular uptake or to an increase in its intracellular deiodination to iodocompounds different than T3. Previous studies have shown that 12 h of LiCl-treatment also decreases T4 tissue concentration, whereas the activity of the 50 DII is already increased (Eravci et al., 2000). Our data estimate that short-term (within 1 day) LiCl-treatment does not alter the intracellular availability of the active form of TH [T3(T4)], although the cytoplasmic T4 levels are lower in adult rat brain, through an increase of the local conversion of T4 to T3. The present data are important in characterizing the intracellular thyroidal status of adult rat brain after shortterm of LiCl-treatment, since it has been shown that 70% of
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the intracellular T3 levels in adult rat brain are derived from local deiodination of T4 (Crantz and Larsen, 1980; Crantz et al., 1982). Furthermore, the short-term LiCl-induced decrease of the cytoplasmic levels of T4 could lead in cellular hypothyroidism after chronic administration of lithium. In agreement with this hypothesis are previous reports showing that the activity of 50 DII is also increased after long-term LiCl-treatment in adult rat brain (Baumgartner et al., 1994); an observation also common in this tissue under hypothyroidism (Leonard et al., 1981; Visser et al., 1983). Additionally, this study shows that the previously observed increase of the nuclear T3 Bmax (Bolaris et al., 1995) is accompanied by alterations in the expression pattern of TR isoforms in adult rat cerebral tissue after shortterm lithium treatment. That is, the present data revealed inversely proportional changes between the nuclear T3 Bmax and the mRNA levels of the TRa2 variant (non-T3 binding isoform of TRs) (# 38%) 24 h after LiCl administration. In contrast, the relative expression of the TRa1 (T3 binding isoform) was increased (" 12%), while no changes have been observed in the relative expression of the other T3 binding TR isoform (TRb1) expressed in adult rat brain at this interval of LiCl-treatment. In order to examine how early the above effects of short-term LiCl-treatment are observed, the relative expression of all TR isoforms was also examined 4 h after LiCl administration. At that interval no changes were observed in the expression levels of the T3 binding TR isoforms (TRa1 and TRb1); however, the TRa2 mRNA levels were decreased (# 16%). That effect was also inversely proportional to the observed increase in the total in vitro T3 binding to cerebral nuclei from adult rats treated with lithium for 4 h (Bolaris et al., 1995). It is noticeable that TR isoforms are differentially affected at different intervals of short-term LiCl-treatment in adult rat cerebral hemispheres. This may suggest that the expression of these isoforms responds differentially to intracellular events brought about by short-term LiCltreatment such as several intracellular signalling pathways (i.e. the cAMP pathway, the phosphoinositol pathway and the protein kinase C pathway), which have been reported to alter by lithium (Brunello, 2004). Alternatively, lithium could affect the TR expression pattern directly in the transcriptional level by affecting the expression and/or the DNA binding capacity of other transcriptional factors regulating the expression of the genes coding for TRs; the AP1 binding on DNA is enhanced by short-term LiCltreatment (Ausgari et al., 1998). It has been suggested that long-term therapeutic efficacy of lithium on bipolar disorder may involve different effects of this drug at the cellular (signalling transduction pathways affected by lithium) and genomic level from its effects after short-term administration (Brunello, 2004). In deed, it has been reported that long-term treatment with lithium increases the TRa2 mRNA levels (Hahn et al., 1999) in adult rat cerebral cortex and hippocampus whereas the
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present data show that short-term LiCl-treatment decreases the TRa2 relative expression in the cerebral hemispheres, indicating thus a physiological relevance for this TR isoform in adult rat brain’s response to lithium treatment. However, it remains to be determined whether these effects of lithium on TR expression and specifically on TRa2 mRNA levels, are related to its therapeutic action in the treatment of bipolar disorder. Although the RNA processing of the TRa1 and TRa2 mRNA is very similar, the expression levels of these two isoforms (derived from a common precursor RNA) are differentially regulated, at least by short-term (within 1 day) LiCl administration, either prior to or after RNA splicing. Thus, it would be very important to delineate if the above changes in the TRa1 and TRa2 mRNA levels are due to changes in the transcriptional activity of the c-erbAa gene or due to alterations in the alternative splicing pattern of this gene (post-transcriptionally) or both. In support to our results, it has been reported that TRa2 is not under regulatory control like the other TR isoforms (Koenig et al., 1988; Lazar et al., 1988; Mitsuhashi and Nikodem, 1989; Hodin et al., 1990). Our results indicate that the regulation of the expression of the non-T3 binding variant of TRs (TRa2) could be relevant in the determination of the maximal number of the nuclear T3 binding sites (i.e. the T3 binding TR isoforms bound on DNA). In support to this hypothesis there are previous reports, which indicate that TRa2 has negative effect on the T3-dependent gene expression due to competitive substitution with the T3 binding TR isoforms on the same TREs in chromatin (Katz and Lazar, 1993); its negative efficacy however, is less potent than the unliganded T3 binding isoforms (Yang et al., 1996; Tagami et al., 1998). Consequently, the LiCl-induced reduction of the TRa2 mRNA levels could cause release of DNA binding sites available for the T3 binding TR isoforms – which were found up-regulated (Fig. 1) – leading thus to the observed increase in the T3 nuclear Bmax (Bolaris et al., 1995). Further, this substitution could promote induction of the T3-dependent transcriptional activity of specific T3 target-genes after short-term lithium treatment in the presence of euthyroid intracellular levels of T3, as they were determined at the cytoplasmic and synaptosomal fraction. In support to the above hypothesis, previous reports have shown that lithium affects the expression of the immediately early genes coding for the c-fos and c-jun proteins (Miller and Mathe, 1997; Ausgari et al., 1998), which belong to the family of the AP1 transcription factors, suggesting thus that the administration of this drug could affect not only the T3-dependent gene expression but also the transcriptional activity of genes that are under the control of other transcription factors such as AP1. However, the resolution of many of the issues raised in this study depends upon the determination whether the above described alterations in the TRa2 mRNA levels is reflected into changes in the respective protein in the nucleus.
Acknowledgements The authors gratefully acknowledge Professor W.W. Chin (Division of Genetics, Department of Medicine, Harvard Medical School) for his generous donation of the full-length cDNAs coding for the TRa1, TRa2 and TRb2 isoforms and Professor P. Stylianopoulou (Department of Public Health, University of Athens) for her kind gift of the GAPDH fulllength cDNA. This work was supported by the Hellenic Ministry of Development, General Secretariat for Research and Technology (PENED95, grant No. ED/1611).
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Acute LiCl-treatment affects the cytoplasmic T4 availability and the expression pattern of thyroid hormone receptors in adult rat cerebral hemispheres Caterina Constantinou, Stamatis Bolaris1, Theony Valcana, Marigoula Margarity* Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras 265 00, Greece Received 6 October 2004; accepted 22 November 2004 Available online 7 January 2005
Abstract We have previously reported that short-term LiCl-treatment affects the kinetic characteristics of thyroid hormone binding in adult rat brain (Bolaris, S., Margarity, M., Valcana, T., 1995. Effects of LiCl on triiodothyronine (T3) binding to nuclei from rat cerebral hemispheres. Biol. Psychiatry 37, 106–111); however, the mechanism underlying the above effects of LiCl administration is yet to be determined. In this study, the effects of lithium within one day after its administration (5 mmol/kg BW) on the relative expression of thyroid hormone receptor isoforms and on the cytoplasmic and synaptosomal thyroid hormone availability in adult rat cerebral hemispheres were examined. Although short-term LiCl-treatment did not affect the levels of triiodothyronine either in the synaptosomal or in the cytoplasmic fraction 24 h after LiCl administration, the cytoplasmic availability of thyroxin was lower. In addition, 24 h after the administration of lithium the mRNA levels of the TRa1 isoform (T3 binding) increased while the relative expression of the TRa2 variant (non-T3 binding) was decreased. Notably, the decrease of the TRa2 mRNA levels was also observed 4 h after LiCl administration. The expression levels of the TRb1 isoform were unaffected in any interval examined. The present study suggests that short-term lithium treatment regulates the relative expression of TRs in an isoform-specific manner and affects the cytoplasmic availability of thyroxin in adult rat brain. # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Lithium (LiCl); Adult rat brain; Thyroid hormone receptors (TR); Triiodothyronine (T3); Thyroxin (T4); Thyroxine availability
1. Introduction Bipolar disorder (manic-depressive illness) is a common psychiatric condition characterized by cycling episodes of mania and severe depression; however, the pathophysiological mechanism(s) underlying bipolar disorder is not completely understood (Goodwin and Jamison, 1990) although molecular genetic studies of this disorder have been proceeded (Kato, 2001). The efficacy of lithium in the treatment of bipolar disorder is well established (Cade, 1949; Frost and Messiha, 1983) and although many cellular effects of lithium have been described, the basis of its * Corresponding author. Tel.: +30 2610 997430; fax: +30 2610 969273. E-mail address: [email protected] (M. Margarity). 1 Present address: Department of Human Reproduction, ‘‘Helena Venizelou’’ Hospital, 2 Venizelou Sqr. 115 21 Athens, Greece.
therapeutic action is unknown (Lenox et al., 1998). Numerous studies have put forward several theories regarding the mechanism(s) underling LiCl therapeutic action, ranging from ion transport to gene expression. Early research focused on LiCl-dependent modulation of presynaptic events and on its effects in postsynaptic intracellular phenomena, involving mainly the cAMP and the phosphoinositide signaling transduction pathways (Brunello, 2004). Recently, it has been shown that lithium treatment confers changes in gene expression (Ausgari et al., 1998) that may account for its therapeutic efficacy in bipolar disorder. A strong correlation between the long-term LiCltreatment and the thyroid hormones (TH) has been well established; chronic lithium administration affects the production and release of TH from the thyroid gland (Burrow et al., 1971; Spaulding et al., 1972; Mori et al.,
0168-0102/$ – see front matter # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2004.11.005
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1989) and the peripheral TH metabolism, including brain tissue (Bagchi et al., 1978; St. Germain, 1987; Baumgartner et al., 1994). In addition, a well-documented theory regarding the therapeutic action of lithium in bipolar disorder has already been proposed (Bolaris et al., 1995). Triiodothyronine (T3), the active form of TH, exerts its physiological role, at the cellular level, mainly through binding to specific nuclear receptors; the TRa1, TRa2, TRb1 and TRb2 isoforms (Thompson et al., 1987; Koenig et al., 1988; Mitsuhashi et al., 1988; Hodin et al., 1989). The TRa1, TRb1 and TRb2 bind T3 with high affinity and also bind to thyroid hormone response elements (TREs) on chromatin, regulating transcriptional processes in several target tissues, including adult rat brain. Binding of unliganded TRa1, TRb1 and TRb2 on TREs represses the basal transcription of the positively regulated T3-target genes; the above described repression is reversed upon binding of T3 to its specific nuclear receptors (Brent et al., 1991; Zhang et al., 1991; Baniahmad et al., 1992; Yen et al., 1993; Li et al., 1999; Wu and Koenig, 2000; Ito and Roeder, 2001; Yen, 2001). Additionally, several negatively regulated T3-target genes have been already identified (Katz and Koenig, 1994; Wu and Koenig, 2000); the mechanism through which the unliganded TRs activate basal transcription of these genes remains unknown, although the involvement of corepressors has been proposed (Tagami et al., 1997, 1999). TRa2, an alternate splice product of the TRa1, cannot bind T3 because it lacks an intact ninth heptad hydrophobic repeat in its ligand binding domain and acquires a unique carboxyl-terminal region (Mitsuhashi et al., 1988; Lazar et al., 1988; Koenig et al., 1989; Katz et al., 1992). The physiological role of this non-T3 binding isoform of TRs remains unknown. The regulation of gene expression has been proposed to be one of the potential mechanisms by which lithium exerts its mood-stabilizing effects (Brunello, 2004). Lithium is capable of regulating the expression of a number of genes in rat brain and cultured-cell models (Sivam et al., 1988, 1989; Colin et al., 1991; Wang and Young, 1996; Miller and Mathe, 1997; Feinstein, 1998; Hua et al., 2000); the c-fos and TR genes, encoding for transcription factors, are among the lithium-regulated genes (Miller and Mathe, 1997; Ausgari et al., 1998; Hahn et al., 1999). However, different effects of lithium on c-fos expression in acute, short-term and long-term lithium treatment have been reported (Miller and Mathe, 1997; Ausgari et al., 1998; Moorman and Leslie, 1998); consequently lithium could affect differentially the down-stream of c-fos gene expression, promoting the cellular adaptation in critical neuronal circuits that lead to mood stabilization. As mentioned above, long-term administration of lithium affects the expression pattern of the thyroid hormone receptors (TR) in several brain areas of adult rats (Hahn et al., 1999). In addition, our previous study has shown an increase of the total nuclear T3 binding after long-term administration of LiCl in adult rat cerebral tissue (Bolaris
et al., 1995). However, it is important to delineate whether the above described effects of lithium on the TH mechanism of action can be also observed following acute administration of this mood stabilizer; this will help to elucidate whether the T3-dependent gene expression is acute responded to lithium. We have previously reported that short-term (24 h) LiCl-treatment increased the in vitro total T3 binding in adult rat cerebral nuclei; an effect that was attributed to an increase of the maximal number of the nuclear T3 binding sites (Bmax), whereas the binding affinity was unaffected (Bolaris et al., 1995). The object of this study was to investigate whether the changes in the nuclear T3 Bmax in adult rat cerebral tissue 24 h after LiCl administration correlate with changes in the expression pattern of the TR isoforms. The cytoplasmic conversion of thyroxin (T4) to T3 and the synaptosomal availability of TH in adult rat cerebral hemispheres were also examined, in order to evaluate the thyroidal status of this tissue after shortterm treatment with lithium.
2. Materials and methods 2.1. Animals and treatments Adult Wistar rats (40 days) of both sexes were bred in our laboratory, were housed 4 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 am to 7 pm; dark period: 7 pm to 7 am; at 22 1 8C) for at least 1–2 weeks prior to LiCl-treatment and until sacrifice. Groups of experimental animals received an intraperitoneal injection of LiCl [5 mmol/kg of body weight (BW)] at 4 and 24 h prior to sacrifice. In all groups, the control animals were injected with an equal volume of 0.9% NaCl. For the study of the in vivo cytoplasmic thyroxin deiodination 24 h LiCl-treated and control animals were injected intraperitoneally with trace 125I-T4 (5 ng/100 g BW; 4400 Ci/mmol, NEN) 4 h prior to sacrifice. After sacrifice, brains were rapidly removed in a sterile cooled glass plate and the cerebral hemispheres were isolated and weighed. For the determination of synaptosomal and cytoplasmic thyroid hormone levels fresh cerebral hemisphere tissue was used. For the mRNA isolation, tissue samples were frozen in liquid N2 until assayed by Northern analysis within 2 weeks. Plasma T3 and T4 levels were determined with specific radioimmunoassay kits (T3-RIA and T4-RIA) from the Hellenic Center of Natural Research, ‘‘Demokritos’’. 2.2. 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 (1992) with slight modifications. Briefly, the cerebral hemispheres
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(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 8C, pH 7.4) and was centrifuged at 34,000 g for 50 min. The crude synaptosomal fraction was banded between the 0.32 and 1.20 M sucrose layers and was carefully removed by suction at 4 8C. 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 composed of 8 ml of 0.85 M and 8 ml of 0.32 M sucrose (4 8C, 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 8C 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 (4 8C, pH 7.4). 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 ml) 10 ml 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 (1976), using bovine serum albumin as standard. 2.3. Identification of cytoplasmic iodothyronines For the study of the in vivo identification of cytoplasmic iodocompounds, 24 h LiCl-treated and control animals were injected intraperitoneally with trace 125I-T4 (5 ng/100 g BW; 4400 Ci/mmol, NEN) 4 h prior to sacrifice. Cerebral hemispheres from two animals were dissected (4 8C), weighed and homogenized in 0.32 M cold sucrose solution (20% w/v, glass-teflon homogenizer, 1000 rpm, 4 8C). After sampling in duplicate for the total radioactivity, the homogenate was centrifuged at 1000 g for 10 min at 4 8C. The supernatant, after addition of dithiothreitol to a final concentration of 5 mM, was immediately centrifuged for 90 min at 100,000 g at 4 8C. The supernatant of this centrifugation (cytoplasmic fraction) was sampled in duplicate and each sample was used for the determination of protein and the distribution of 125I-T3 and 125I-T4 among total radioactivity. For this purpose, the cytoplasmic fraction was treated with 95% ethanol until 85% or more of the radioactivity contained was extracted. These extracts were evaporated to dryness under nitrogen in a 37 8C water bath in the dark. The residue of these samples was dissolved in a mixture of methanol/concentrated NH3 (3:1, v/v). The iodocompounds of these extracts were identified and quantified by thin-layer chromatography (TLC) as pre-
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viously described by Heider and Bronk (1956). The protein content of the cytoplasmic fraction was determined by the method of Lowry et al. (1951). 2.4. Northern blot hybridization The relative expression of each thyroid hormone receptor isoform was determined by Northern blot hybridization. PolyA+RNA was extracted from 1 g of cerebral hemispheres from LiCl-treated and control animals utilizing the Poly(A)PureTM kit (Ambion). In each experiment three different polyA+RNA preparations from LiCl-treated and control animals were analyzed by Northern blot hybridization twice in duplicate for each TR isoform mRNA as described previously by Sambrook et al. (1989). Briefly, after electrophoresis of polyA+RNA samples in 1% agarose/ formaldehyde gel, the electrophoretic-separated mRNA was transferred overnight to positively charged nylon membranes (NEN) and subsequently cross-linked by baking the membranes at 80 8C for 2 h. The blots were prehybridized at 68 8C in UltraHyb hybridization solution (Ambion) for 2 h. Hybridization was performed overnight at 62 8C for TRa1 isoform, 68 8C for TRa2 isoform and 65 8C for TRb1 and TRb2 isoforms with 3 106 cpm/ml of random labeled probes arising by digestion of the TRa1, TRa2 and TRb2 fulllength cDNAs, with restriction enzymes (a1 specific probe: EcoRI and XbaI in a1 cDNA, a2 specific probe: EcoRI and XhoI in a2 cDNA, b1/b2 common probe and b2 specific probe: XbaI in b2 cDNA). Blots were washed according to the instructions of NEN for the particular membrane used and then were exposed immediately to the Phosphorimager cassette (Molecular Dynamics) for 24 h and quantified by computer-assisted densitometry. PolyA+RNA recovery was determined by rehybridization of the membranes with the glyceraldhehyde-3-phosphate-dehydrogenase (GAPDH) probe and all values were normalized to the GAPDH signal. 2.5. Statistics Results were expressed as the mean S.E., analyzed statistically by performing one-way completely randomized analysis of variance (ANOVA) with the P < 0.05 as the significance cutoff point. One-way analysis of variance with a Bonferroni correction for multiple comparisons was performed for the effect of short-term LiCl-treatment on the relative expression of all TR isoforms and on T3 synaptosomal levels.
3. Results 3.1. The effects of short-time LiCl-treatment on the thyroid hormone synaptosomal and cytoplasmic levels Administration of LiCl for 4 and 24 h had no significant effect on plasma T4 and T3 levels (T4: 33.4 2 ng/ml; T3:
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0.734 0.1 ng/ml). These findings are in agreement with our previous data (Bolaris et al., 1995). The determination of the iodothyronines (125I-T3 and 125 I-T4), following in vivo 125I-T4 injection in the total cytoplasmic fraction of adult rat cerebral hemispheres is presented in Table 1. These data show that 24 h of LiCltreatment had no effect on the 125I-T3 levels (125I-T3: 53.9 8.4 106 ng/mg protein) resulting from the local deiodination of 125I-T4 [T3(T4)]; however, the levels of 125I-T4 were decreased (47%) in the same intracellular fraction (Control 125I-T4: 108.5 22.5 106 ng/mg protein; LiCl-treated 125I-T4: 57.4 3.1 106 ng/mg protein, P < 0.01). Short-term LiCl-treatment (5 mmol/kg BW, 4 and 24 h prior to sacrifice) had no effect on the synaptosomal levels (0.473 0.05 ng/mg protein) of the active form of thyroid hormones (T3). In agreement with data from the literature (Sarkar and Ray, 1994) the T4 synaptosomal levels remained undetectable in all control and LiCl-treated animals examined in this study. 3.2. The effects of short-time LiCl-treatment on the relative expression of all thyroid hormone receptor isoforms in adult rat cerebral hemispheres Northern blot hybridization was employed in order to study the effect of short-term LiCl-treatment on the relative expression of TRs in cerebral hemispheres from control and LiCl-treated animals (5 mmol/kg BW, 24 and 4 h prior to sacrifice). Our results show that 24 h of LiCl-treatment increased the TRa1 (T3 binding) relative expression (12%) (Fig. 1), while the TRa2 variant’s (non-T3 binding) mRNA levels were decreased (38%) (Fig. 2). A reduction of the TRa2 relative expression was also observed 4 h (16%) after LiCl administration, whereas the TRa1 mRNA levels were unaffected at this interval. In addition, the expression levels of the TRb1 isoform (T3 binding) were not affected in any interval examined within 24 h after LiCl administration. Moreover, in agreement with results of previous studies (Hodin et al., 1989; Strait et al., 1990) the TRb2 isoform could not be detected by Northern analysis in cerebral
Table 1 Effects of 24 h LiCl-treatment on the cytoplasmic T4 deiodination in cerebral hemispheres of adult rats injected with 125I-T4
Control LiCl-treated
125
125
125
I-T4 (106ng/mg protein)
I-T3( I-T4) (106ng/mg protein)
108.5 22.5 57.4 3.1* (# 47%)
53.9 8.4 59.1 13
The LiCl-treated animals received LiCl (5 mmol/kg BW) 24 h prior to sacrifice. Control animals received an equal volume of 0.9% NaCl. All control and LiCl-treated animals received also trace of 125I-T4 (5 ng/100 g BW; spec. activ. 4400 Ci/mmol, NEN) 4 h prior to sacrifice. Numbers represent means S.E. of four preparations (one animal per preparation) assayed in triplicate. * Statistically different from control (P < 0.01, analysis of variance).
Fig. 1. (A) (Top) Northern blot analysis of TRa1 variant from adult rat cerebral hemispheres. 5 mg of polyA+RNA preparations from cerebral hemispheres of control and LiCl-treated (5 mmol/kg BW, 4 and 24 h prior to sacrifice) adult rats were applied in 1% agarose/formaldehyde gel. Lanes 1, 2: control; Lanes 3, 4: LiCl-treated, 4 h; Lanes 5, 6: LiCl-treated, 24 h. (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) Effect of short-term LiCl-treatment on the relative expression of the TRa1 mRNA levels in adult rat cerebral hemispheres. Northern blots from five separate experiments quantified as described under Section 2. All data are expressed as arbitrary units (AU) per mg polyA+RNA and all values represent means S.E. of five different experiments. *Statistically different from control (P < 0.05, analysis of variance).
hemispheres of all control or LiCl-treated animals (data not shown).
4. Discussion The regulation of the T3-dependent gene expression is directly dependent upon the cytoplasmic TH availability and the density of the T3 nuclear receptors. The purpose of this study was the determination of the intracellular thyroidal status of adult rat brain, as well as to examine whether there is a correlation between the observed increase of the number of the nuclear T3 receptors (Bolaris et al., 1995) and the expression pattern of the TR isoforms in adult rat cerebral hemispheres after short-term (within 1 day) LiCl-treatment. Our results show that the cytoplasmic levels of T3(T4) were not affected; however, a reduction (47%) in the
C. Constantinou et al. / Neuroscience Research 51 (2005) 235–241
Fig. 2. (A) (Top) Northern blot analysis of TRa2 variant from adult rat cerebral hemispheres. 5 mg of polyA+RNA preparations from cerebral hemispheres of control and LiCl-treated (5 mmol/kg BW, 4 and 24 h prior to sacrifice) adult rats were applied in 1% agarose/formaldehyde gel. Lanes 1, 2: Control; Lanes 3, 4: LiCl-treated, 4 h; Lanes 5, 6: LiCl-treated, 24 h. (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) Effect of short-term LiCl-treatment on the relative expression of the TRa2 mRNA levels in adult rat cerebral hemispheres. Northern blots from five separate experiments quantified as described under Section 2. All data are expressed as arbitrary units (AU) per mg polyA+RNA and all values represent means S.E. of five different experiments. *Statistically different from control (P < 0.05, analysis of variance).
cytoplasmic levels of T4 was observed in the absence of any alteration in the plasma and synaptosomal TH levels. The above LiCl-induced decrease of cytoplasmic T4 levels could be attributed to a reduction in the T4 intracellular uptake or to an increase in its intracellular deiodination to iodocompounds different than T3. Previous studies have shown that 12 h of LiCl-treatment also decreases T4 tissue concentration, whereas the activity of the 50 DII is already increased (Eravci et al., 2000). Our data estimate that short-term (within 1 day) LiCl-treatment does not alter the intracellular availability of the active form of TH [T3(T4)], although the cytoplasmic T4 levels are lower in adult rat brain, through an increase of the local conversion of T4 to T3. The present data are important in characterizing the intracellular thyroidal status of adult rat brain after shortterm of LiCl-treatment, since it has been shown that 70% of
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the intracellular T3 levels in adult rat brain are derived from local deiodination of T4 (Crantz and Larsen, 1980; Crantz et al., 1982). Furthermore, the short-term LiCl-induced decrease of the cytoplasmic levels of T4 could lead in cellular hypothyroidism after chronic administration of lithium. In agreement with this hypothesis are previous reports showing that the activity of 50 DII is also increased after long-term LiCl-treatment in adult rat brain (Baumgartner et al., 1994); an observation also common in this tissue under hypothyroidism (Leonard et al., 1981; Visser et al., 1983). Additionally, this study shows that the previously observed increase of the nuclear T3 Bmax (Bolaris et al., 1995) is accompanied by alterations in the expression pattern of TR isoforms in adult rat cerebral tissue after shortterm lithium treatment. That is, the present data revealed inversely proportional changes between the nuclear T3 Bmax and the mRNA levels of the TRa2 variant (non-T3 binding isoform of TRs) (# 38%) 24 h after LiCl administration. In contrast, the relative expression of the TRa1 (T3 binding isoform) was increased (" 12%), while no changes have been observed in the relative expression of the other T3 binding TR isoform (TRb1) expressed in adult rat brain at this interval of LiCl-treatment. In order to examine how early the above effects of short-term LiCl-treatment are observed, the relative expression of all TR isoforms was also examined 4 h after LiCl administration. At that interval no changes were observed in the expression levels of the T3 binding TR isoforms (TRa1 and TRb1); however, the TRa2 mRNA levels were decreased (# 16%). That effect was also inversely proportional to the observed increase in the total in vitro T3 binding to cerebral nuclei from adult rats treated with lithium for 4 h (Bolaris et al., 1995). It is noticeable that TR isoforms are differentially affected at different intervals of short-term LiCl-treatment in adult rat cerebral hemispheres. This may suggest that the expression of these isoforms responds differentially to intracellular events brought about by short-term LiCltreatment such as several intracellular signalling pathways (i.e. the cAMP pathway, the phosphoinositol pathway and the protein kinase C pathway), which have been reported to alter by lithium (Brunello, 2004). Alternatively, lithium could affect the TR expression pattern directly in the transcriptional level by affecting the expression and/or the DNA binding capacity of other transcriptional factors regulating the expression of the genes coding for TRs; the AP1 binding on DNA is enhanced by short-term LiCltreatment (Ausgari et al., 1998). It has been suggested that long-term therapeutic efficacy of lithium on bipolar disorder may involve different effects of this drug at the cellular (signalling transduction pathways affected by lithium) and genomic level from its effects after short-term administration (Brunello, 2004). In deed, it has been reported that long-term treatment with lithium increases the TRa2 mRNA levels (Hahn et al., 1999) in adult rat cerebral cortex and hippocampus whereas the
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present data show that short-term LiCl-treatment decreases the TRa2 relative expression in the cerebral hemispheres, indicating thus a physiological relevance for this TR isoform in adult rat brain’s response to lithium treatment. However, it remains to be determined whether these effects of lithium on TR expression and specifically on TRa2 mRNA levels, are related to its therapeutic action in the treatment of bipolar disorder. Although the RNA processing of the TRa1 and TRa2 mRNA is very similar, the expression levels of these two isoforms (derived from a common precursor RNA) are differentially regulated, at least by short-term (within 1 day) LiCl administration, either prior to or after RNA splicing. Thus, it would be very important to delineate if the above changes in the TRa1 and TRa2 mRNA levels are due to changes in the transcriptional activity of the c-erbAa gene or due to alterations in the alternative splicing pattern of this gene (post-transcriptionally) or both. In support to our results, it has been reported that TRa2 is not under regulatory control like the other TR isoforms (Koenig et al., 1988; Lazar et al., 1988; Mitsuhashi and Nikodem, 1989; Hodin et al., 1990). Our results indicate that the regulation of the expression of the non-T3 binding variant of TRs (TRa2) could be relevant in the determination of the maximal number of the nuclear T3 binding sites (i.e. the T3 binding TR isoforms bound on DNA). In support to this hypothesis there are previous reports, which indicate that TRa2 has negative effect on the T3-dependent gene expression due to competitive substitution with the T3 binding TR isoforms on the same TREs in chromatin (Katz and Lazar, 1993); its negative efficacy however, is less potent than the unliganded T3 binding isoforms (Yang et al., 1996; Tagami et al., 1998). Consequently, the LiCl-induced reduction of the TRa2 mRNA levels could cause release of DNA binding sites available for the T3 binding TR isoforms – which were found up-regulated (Fig. 1) – leading thus to the observed increase in the T3 nuclear Bmax (Bolaris et al., 1995). Further, this substitution could promote induction of the T3-dependent transcriptional activity of specific T3 target-genes after short-term lithium treatment in the presence of euthyroid intracellular levels of T3, as they were determined at the cytoplasmic and synaptosomal fraction. In support to the above hypothesis, previous reports have shown that lithium affects the expression of the immediately early genes coding for the c-fos and c-jun proteins (Miller and Mathe, 1997; Ausgari et al., 1998), which belong to the family of the AP1 transcription factors, suggesting thus that the administration of this drug could affect not only the T3-dependent gene expression but also the transcriptional activity of genes that are under the control of other transcription factors such as AP1. However, the resolution of many of the issues raised in this study depends upon the determination whether the above described alterations in the TRa2 mRNA levels is reflected into changes in the respective protein in the nucleus.
Acknowledgements The authors gratefully acknowledge Professor W.W. Chin (Division of Genetics, Department of Medicine, Harvard Medical School) for his generous donation of the full-length cDNAs coding for the TRa1, TRa2 and TRb2 isoforms and Professor P. Stylianopoulou (Department of Public Health, University of Athens) for her kind gift of the GAPDH fulllength cDNA. This work was supported by the Hellenic Ministry of Development, General Secretariat for Research and Technology (PENED95, grant No. ED/1611).
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