Download Region-specific effects of hypothyroidism on the relative expression

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. Chin (Division of Genetics, Department of Medicine, Brigham and
Women’s Hospital, and Howard Hughes Medical Institute,
Harvard Medical School) for his generous donation of the
full-length cDNAs coding for the TRα1, TRα2 and TRβ2
isoforms and Prof. P. Stylianopoulou (Department of Public Health, University of Athens) for her kind gift of the
GαPDH full-length cDNA. This work was supported by the
Hellenic Ministry of Development, General Office of Research and Technology (Grant No. 95ED 1611 and Grant
No. 99ED 106).
References
1. Lazar MA: Thyroid hormone receptors: multiple forms, multiple possibilities. Endocrinol Rev 14: 240–279, 1993
2. Thompson CC, Weinberger C, Lebo R, Evans RM: Identification of a
novel thyroid hormone receptor expressed in the mammalian central
nervous system. Science 237: 1610–1614, 1987
3. Koenig RJ, Warne RL, Brent GA, Harney JW, Larsen PR, Moore DD:
Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor. Proc Natl Acad Sci USA 85: 5031–5035, 1988
4. Mitsuhashi T, Tennyson GE, Nikodem VM: Alternative splicing generates messages encoding rat c-erbA proteins that do not bind thyroid
hormone. Proc Natl Acad Sci USA 85: 5804–5808, 1988
5. Hodin RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen PR,
Moore DD, Chin WW: Identification of thyroid hormone receptor that
is pituitary-specific. Science 249: 76–79, 1989
6. Koenig RJ, Lazar MA, Hodin RA, Brent GA, Larsen PR, Chin WW,
Moore DD: Inhibition of thyroid hormone action by a non-hormone
binding c-erbA protein generated by alternative RNA splicing. Nature
337: 659–661, 1989
7. Yen PM: Physiological and molecular basis of thyroid hormone action.
Physiol Rev 81: 1097–1142, 2001
8. Chen JD, Evans RM: A transcriptional corepressor that interact with
nuclear receptors. Nature 377: 454–457, 1995
9. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R,
Ryan A, Kamel Y, Soderstrom M, Glass CK, Rosenfeld MG: Ligandindependent repression by thyroid hormone receptor mediated by a nuclear co-repressor. Nature 377: 397–404, 1995
10. Seol WM, Choi MS, Moore DD: Isolation of proteins that interact specifically with the retinoid X receptor: Two novel orphan receptors. Mol
Endocrinol 9: 72–85, 1995
11. Sande S, Privalski ML: Identification of TRACs (T3 receptorassociating), a family of cofactors that associate with, and modulate the
activity of nuclear hormone receptors. Mol Endocrinol 10: 813–825,
1999
12. Yen PM, Spanjaard RA, Suguwara A, Darling DS, Nguyen VP, Chin
WW: Orientation and spacing of half-sites differentially affect T3 receptor (TR) monomer, homodimer and heterodimer binding to thyroid hormone response elements (TREs). Endocrinol J 1: 461–466,
1993
13. Li Q, Sachs L, Shi Y-B, Wolfe AP: Modification of chromatin structure
by thyroid hormone receptor. Trends Endocrinol Metab 10: 157–164,
1999
14. Wu Y, Koenig RJ: Gene regulation by thyroid hormone. Trends Endocrinol Metab 11: 207–211, 2000
15. Ito M, Roeder RG: The TRAP/SMCC/mediator complex and thyroid
hormone receptor function. Trends Endocrinol Metab 12: 127–134,
2001
16. Katz D, Berrodin TJ, Lazar MA: The unique c-termini of the thyroid
hormone receptor variant c-ErbA alpha 2 and thyroid hormone receptor
alpha 1 mediate different DNA-binding and heterodimerization properties. Mol Endocrinol 6: 805–814, 1992
17. Yang YZ, Burgos-Trinidad M, Wu Y, Koenig RJ: Thyroid hormone
receptor variant alpha 2. Role of the ninth heptad in DNA binding,
heterodimerization with retinoic X receptors, and dominant negative
activity. J Biol Chem 271: 28235–28242, 1996
18. Tagami J, Kopp P, Johnson W, Arserven K, Jameson JL: The thyroid
hormone receptor variant α2 is a weak antagonist because it is deficient
in interactions with nuclear co repressors. Endocrinology 139: 2535–
2544, 1998
19. Sakurai A, Nakai A, DeGrout LJ: Expression of three forms of thyroid
hormone receptor in human tissues. Mol Endocrinol 3: 392–399, 1989
20. Strait KA, Schwartz HL, Perez-Castillo A, Oppenheimer JH: Relationship of c-erbA mRNA content to tissue triiodothyronine binding capacity and function in developing and adult rats. J Biol Chem 265:
10514–10521, 1990
21. Forrest D, Hallbook F, Persson H, Vennström B: Distinct functions for
thyroid hormone receptors α and β in brain development indicated
by differential expression of receptor genes. J EMBO 10: 269–275,
1991
22. Clerget-Froidevaux MS, Seugnet I, Demeneix BA: Thyroid status coregulates thyroid hormone receptor and co-modulator genes specifically
in the hypothalamus. FEBS Lett 569: 341–345, 2004
23. Mitsuhashi T, Nikodem VM: Regulation of expression of the alternative
mRNAs of the rat α-thyroid hormone receptor gene. J Biol Chem 264:
8900–8904, 1989
24. Bradley DJ, Young WS, Weinberger C: Differential expression of α and
β thyroid hormone receptor genes in rat brain and pituitary. Proc Natl
Acad Sci USA 86: 7250–7254, 1989
25. Gauthier K, Chassande O, Plateroti M, Roux J-P, Legrand C, Pain B,
Rousset B, Weiss R, Trouillas J, Samarut J: Different functions for
the thyroid hormone receptors TRα and TRβ in the control of thyroid
hormone production and post-natal development. J EMBO 18: 623–631,
1999
26. Forrest D, Vennström B: Functions of thyroid hormone receptors in
mice. Thyroid 10: 41–51, 2000
27. Flamant F, Samarut J: Thyroid hormone receptors: lessons from knockout and knock-in mutant mice. Trends Enocrinol Metab 14: 85–90, 2003
28. Morte B, Manzano J, Scanlan TS, Vennström B, Bernal J: Deletion of
the thyroid hormone receptor α1 prevents the structural alterations of
the cerebellum induced by hypothyroidism. Proc Natl Acad Sci USA
99: 3985–3989, 2002
29. Manzano J, Morte B, Scanlan TS, Bernal J: Differential effects of triiodothyronine and the thyroid hormone receptor β-specific agonist GC1 on thyroid hormone target genes in the brain. Endocrinology 144:
5480–5487, 2003
30. Hsu J-H, Brent GA: Thyroid hormone receptor gene knockouts. Trends
Endocrinol Metab 9: 103–112, 1998
31. Zhang J, Lazar MA: The mechanism of action of thyroid hormones.
Annu Rev Physiol 62: 439–466, 2000
32. Hashimoto K, Curty FH, Borges PP, Lee CE, Abel ED, Elmquist JK,
Cohen RN, Wondisford FE: An unliganded thyroid hormone receptor
causes severe neurological dysfunctions. Proc Natl Acad Sci USA 98:
3998–4003, 2001
33. Constantinou C, Bolaris S, Valcana T, Margarity M: Acute LiCl treatment affects the cytoplasmic availability and the expression pattern of
thyroid hormone receptors in adult rat cerebral hemispheres. Neurosci
Res 51: 235–241, 2005
100
34. Bolaris S, Constantinou C, Valcana T, Margarity M: Pentylenetetrazoleinduced convulsions affect cellular and molecular parameters of the
mechanism of action of triiodothyronine in adult rat brain. Neuropharmacology 52: 269–275, 2005
35. Kaplan MM: Regulatory influences on iodothyronine deiodination in animal tissue. In: Georg Hennemann (ed). Thyroid Hormone Metabolism.
New York, Marcel Dekker, 1986, pp 231–253
36. Valcana T, Timiras PS: Nuclear triiodothyronine receptors in the developing rat brain. Mol Cell Endocrinol 11: 31–41, 1978
37. Moreira DG, Marassi MP, Correa da Costa VM, Carvalho DP, Rosenthal
D: Effects of ageing and pharmacological hypothyroidism on pituitary–
thyroid axis of Dutch–Miranda and Wistar rats. Exp Gerontol 40: 330–
334, 2005
38. Sarkar PK, Ray AK: A simple biochemical approach to differentiate
synaptosomes and non-synaptic mitochondria from rat brain. Meth Find
Exp Clin Pharmacol 14: 493–497, 1992
39. Bradford MM: A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein – dye
binding. Anal Biochem 72: 248–254, 1976
40. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning – a Laboratory Manual, 2nd ed. Cold Spring Harbor Press, New York,
1989
41. Sarkar PK, Ray AK: Synaptosomal T3 content in cerebral cortex of
adult rat in different thyroidal states. Neuropsychopharmacology 11:
151–155, 1994
42. Eberhardt NL, Valcana T, Timiras PS: Triiodothyronine nuclear receptors: An in vitro comparison of binding of triiodothyronine to nuclei of
adult rat liver, cerebral hemispheres and anterior pituitary. Endocrinology 102: 556–561, 1978
43. Hamada S, Yoshimasha Y: Increases in brain nuclear triiodothyronine
receptors associated with increased triiodothyronine in hyperthyroid and
hypothyroid rats. Endocrinology 112: 207–211, 1983
44. Dratman MB, Crutchfield FL, Axelrod J, Colburn, RW, Nguyen T: Localization of triiodothyronine in nerve ending fractions of rat brain. Proc
Natl Acad Sci USA 73: 941–944, 1976
45. Dratman MB, Crutchfield FL: Synaptosomal [125 I]-triiodothyronine after intravenous [125 I]-thyroxine. Am J Physiol 235: 638–647, 1978
46. Valcana T: The role of triidothyronine (T3 ) receptors in brain development. In: E. Meisami, M.A.B. Brazier (eds). Neural Growth and Differentiation. Raven, New York, 1979, pp 39–58
47. Lazar MA, Hodin RA, Cardona G, Chin WW: Gene expression from the
c-erbAα/Rev-ErbAα genomic locus: Potential regulation of alternative
splicing opposite strand transcription. J Biol Chem 265: 12859–12863,
1990
48. Hastings ML, Milcarek C, Matrincic K, Peterson ML, Munroe SE: Expression of the thyroid hormone receptor gene, erbA-alpha, in B lymphocytes: alternative mRNA processing is independent of differentiation but
correlates with antisense RNA levels. Nucleic Acid Res 25: 4296–4300,
1997
49. Hodin RA, Lazar MA, Chin WW: Differential and tissue-specific regulation of the multiple rat c-erbA messenger RNA species by thyroid
hormone. J Clin Invest 85: 101–105, 1990
50. Chassande O, Fraichard A, Gauthier K, Flamant F, Legrand C, Savatier P,
Laudet V, Samarut J: Identification of transcripts initiated from an internal promoter in the c-erbAα locus that encode inhibitors of retinoic acid
receptor-α and thriiodothyronine receptor activities. Mol Endocrinol 11:
1278–1290, 1997
51. Lazar MA, Hodin RA, Darling DS, Chin WW: A novel member of
the thyroid/steroid hormone receptor family is encoded by the opposite
strand of the rat c-erbA alpha transcriptional unit. Mol Cell Biol 9:
1128–1136, 1989
52. Burgos-Trinidad M, Koenig RJ: Dominant negative activity of thyroid
hormone receptor variant alpha 2 and interaction with nuclear corepressors. Mol Cell Endocrinol 149: 107–114, 1999
53. Katz RW, Koenig RJ: Specificity and mechanism of thyroid hormone
induction from an octamer response element. J Biol Chem 269: 18915–
18920, 1994
54. Katz D, Lazar MA: Dominant negative activity of an endogenous thyroid
hormone receptor variant (α2) is due to competition for binding sites on
target genes. J Biol Chem 268: 20904–20910, 1993