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2 and a Concomitant Overexpression of 1 Yields a Mixed Hypo- and Hyperthyroid Phenotype in Mice
Department of Cell and Molecular Biology (B.V., C.S., A.M., H.G., K.N.), Department of Physiology (C.J., P.T.), Karolinska Institute, S-171 77 Stockholm, Sweden; Department of Human Genetics (Z.W., D.F.), Mount Sinai School of Medicine, New York, New York 10029; and Endocrine Division (J.M.K., C.O.), Department of Internal Medicine, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden
Address all correspondence and requests for reprints to: Dr. Björn Vennström, Department of Cell and Molecular Biology, Box 285, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail: Bjorn.Vennstrom{at}cmb.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß,
which each generate variant proteins. In mammals, the gene
generates, in addition to the normal receptor TR1, a
non-hormone-binding variant TR2 whose exact function is unclear.
Here, we present the phenotype associated with the targeted ablation of
TR2 expression. Selective ablation of TR2 resulted in an
inevitable, concomitant overexpression of TR1. Both TR2 +/- and
-/- mice show a complex phenotype with low levels of free
T3 and free T4, and have inappropriately normal
levels of TSH. The thyroid glands exhibit mild morphological signs of
dysfunction and respond poorly to TSH, suggesting that the genetic
changes affect the ability of the gland to release thyroid hormones.
However, the phenotype of the mutant mice also has features of
hyperthyroidism, including decreased body weight, elevated heart rate,
and a raised body temperature. Furthermore, TR2-/- and TR2+/-
mice are obese and exhibit skeletal alterations, associated with a
late-onset growth retardation. The results thus suggest that the
overexpression of TR1 and the concomitant decrease in TR2
expression lead to a mixed hyper- and hypothyroid phenotype, dependent
on the tissue studied.
The phenotypes suggest that the balance of TR1:TR2 expressed from
the TR gene provides an additional level of tuning the control of
growth and homeostasis in mammalian species.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The functions of TH are mediated by nuclear hormone receptors, which
belong to a family of ligand-dependent transcription factors
(5). The TRs are encoded by two distinct but closely
related genes, TR and TRß (6, 7). The TR gene
generates the TR1 and TR2 isoforms that are identical for the
first 370 amino acids but differ as a consequence of differential
splicing at their C terminus: the last 40 specific residues of TR1
are replaced by 122 amino acids encoded by the TR2-specific exon 10,
the last exon in the TR locus. As a consequence of the C-terminal
change, the TR2 protein is unable to bind TH, and no other ligand
has been identified (8, 9, 10). TR2 binds DNA weakly and
only binds a subset of T3-responsive sites
(T3REs). Furthermore, it dimerizes poorly with
RXR and lacks the activating function domain 2 that interacts with
coactivators (11, 12). Although TR2 is highly conserved
in man, rat, and mouse it appears to be absent in nonmammalian
vertebrates (13, 14).
The TR1 and TR2 RNAs are widely coexpressed early in development
(10) and in adult tissues (8, 15, 16, 17). TR2
expression levels are generally 2- to 10-fold higher than those of
TR1. TR2 has been suggested to exert a suppressive function on
other TRs (18). Suggested mechanisms for suppression
include competition for binding to thyroid hormone response elements
(TREs) on target genes, formation of inactive heterodimers, or
squelching (19, 20, 21). A dominant negative effect of
TR2 has also been reported to occur without binding to certain TREs
(22). Recently, TR2 was described to be only a weak
antagonist of TH action due to its low affinity for several response
elements and its failure to interact with corepressors
(12). Dephosphorylation at the C terminus of TR2 has
been reported to increase its DNA binding affinity
(23).
To understand the role of TRs in development, mice deficient in the
expression of one or several TR isoforms have been developed. We
previously showed (24) that mice deficient in TR1 but
retaining TR2 are fully viable although they exhibit lower heart
rate, lower body temperature, and slower ventricular repolarization as
compared with normal mice.
The TRß-deficient mice (25) have severely impaired
hearing, have high serum levels of TH and TSH, and have goiter
(25, 26, 27). They also exhibit a slightly increased heart
rate that is nonresponsive to T3
(28). Mice lacking all known receptors for TH
(TR1-/-ß-/- mice) are viable, suggesting that the
T3 receptors encoded in the TR and TRß loci
are dispensable for life. These mice exhibit growth retardation, delay
in bone maturation, and poor female reproduction (29).
Studies of heart function and control of body temperature revealed
defects similar to those found in TR1-/- mice (28),
suggesting that TR1 has the primary role in these processes.
Surprisingly, the disruption of one of the first exons on the TR
locus (30), resulting in a lack of TR1 and TR2
expression, showed a dramatic phenotype in which pups die shortly after
weaning unless treated with T3. In these mice the
thyroid gland fails to develop properly, and both bone and small
intestine maturation is delayed. It was suggested that the lethality of
the TR-/- mice was caused by residual expression of short variants
of TR proteins that lack DNA-binding domains. Alternatively, the
TR2 protein could have vitally important functions in mammals, or
the TR2 protein may have compensatory functions that overlap those
of TR1 instead of having the antagonistic role that had been
suggested.
To address this issue, mice with a selective disruption of TR2 were
generated by gene targeting in embryonic stem cells. The mice
overexpress TR1 as an inevitable consequence of the targeting event
and are viable. The levels of free T3
(FT3) and free T4
(FT4) are significantly reduced, and their
thyroid glands show features of dysfunction. The mice also exhibit
other signs of hypothyroidism, such as a retarded growth spurt,
increased fat content, decreased cortical bone dimensions, and
decreased trabecular bone mineral density. TSH serum levels, however,
are inappropriately normal, suggesting an alteration in the
pituitary-thyroid axis. In addition, when compared with wild-type (wt)
mice, the mutants have a higher heart rate and body temperature,
parameters usually associated with elevated TH levels. The results
indicate that the loss of TR2 or the changed balance of
TR1/TR2 perturbs a range of functions, suggesting a role for
TR2 in fine tuning mammalian growth and homeostasis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2 Mutant Mice2 expression, we decided to block
transcription of the TR2-specific exon 10. For this purpose, we
introduced a strong polyadenylation site and transcriptional stop in
the 3'-untranslated region of the TR1 mRNA, thus preventing
transcription into the TR2-specific exon 10. This approach was
necessary, since the splice donor site for exon 10 is located within
exon 9, and its alteration would have generated a mutant TR1 protein
(Fig. 1
). The procedure used also avoided
generation of truncated TR1 proteins and left the overlapping
rev-erbA gene intact (31, 32). This approach was
expected to raise the level of TR1 expression in mutant cells equal
to that found for TR2 in wt tissues. This consequence, however,
cannot be avoided in any exclusive ablation of TR2 expression.
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. The DNA fragments
obtained after the BamHI digestion had the expected size, 23
kb from the wt allele and 13 kb or 9.9 kb from the mutated allele, when
5'-probe or 3'-probe were used, respectively. An internal probe, ex10p,
excluded the random integration of the targeting vector in this clone.
Two other independent digestions of the DNA from the 165 clone
(XbaI, StuI) confirm that homologous
recombination occurred as intended.
Cells from clone 165 were injected into blastocysts of C57BL/6 mice,
and several chimeric mice were generated. Mating of heterozygous
offspring resulted in a non-Mendelian distribution of surviving pups:
in 231 mice genotyped from heterozygote (+/-) intercrosses, we
obtained 33% wt, 56% TR2+/-, and 11% TR2-/- mice. The ratio
between male and female offspring was 1:1. However, in a different
facility, numbers of TR2-/- progeny increased to near Mendelian
ratios: 23% wt, 58% TR2+/-, and 21% TR2-/- (total progeny,
n = 102). This suggested that survival of TR2-/- mice was
susceptible to environmental factors. Female TR2-/- mice generally
failed to conceive, or if pups were born, to rear their offspring. The
+/- and -/- females had extended estrous cycles: 4.9 ±
0.2 d (n = 25) and 5.4 ± 0.1 d (n = 20),
respectively, as compared with wt, 4.3 ± 0.2 d (n = 22)
(P < 0.05). Thus, the mutation impaired
reproduction.
Abrogation of TR2 RNA Expression
Next, we verified that expression of TR2 RNA in the mutant mice
was abrogated. RNA from different tissues of wt, TR2+/-, and
TR2-/- mice were analyzed. Northern blots of brain RNA (Fig. 2A) showed the different wt and mutant
TR RNAs in wt, TR2+/-, and TR2-/- mice after hybridization
with probes that recognized both TR1 and TR2, or with specific
probes that recognize either of the specific RNAs isoforms. As
expected, no TR2 RNA was found in the TR2-/- mice, as the wt
2.6-kb band was undetectable in the -/- samples analyzed. The TR1
RNA from the targeted allele, TR11, was shorter (2.2 kb) than the wt
RNA (5.8 kb) as a consequence of the targeting event and was
present in both TR2+/- and -/- mice. In the latter mice the
normal TR1 RNA was undetectable. TRß1 and rev-erbA levels of
expressions were unaltered in the TR2+/- and -/- mice (Fig. 2A, left panel).
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2 expression, we performed RT-PCR with RNA
obtained from 13–15-d-old embryos (E13–E15) and several tissues from
newborn and adult mice: heart, liver, and brain. Figure 2B (lanes 1–4,
lower panel) shows that primers specific for TR2 failed
to amplify any transcript in the homozygous mice, whereas samples from
heterozygous and wt animals scored positive as expected (lanes 5–12,
lower panel). The primers common for wt TR1 and TR11
sequences generated the expected amplified fragments (lanes 1–12,
upper panel). To control for any possible DNA contamination,
the RNA samples were used in amplification experiments in which reverse
transcriptase had been omitted, and no DNA fragments were detected
(Fig. 2C, lanes 5–8 and 13–16). We therefore conclude that expression
of the TR2 product had been efficiently abrogated, as no detectable
expression of TR2 could be observed in any of the different tissues
analyzed from the mutant mice.
Overexpression of TR1
Because the alteration in the TR locus was expected to
increase the expression of TR1, we determined TR1 RNA and protein
levels in different tissues. The novel TR11 transcript was expressed
at higher levels than the wt TR1 RNA in brain tissue from
heterozygous (3- to 5-fold) and homozygous animals (6- to 10-fold), as
revealed by phosphoimager quantification (Fig. 2A). The overexpression
was detected in all the tissues analyzed: brain, pituitary (Fig. 3A), eye, white adipose tissue, heart,
and muscle, with values that ranged from 3- to 10-fold (data not
shown). No significant alteration in the expression of TRß2 RNA was
observed (Fig. 3A).
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11 RNA gave
rise to an overproduction of functional TR1 receptor in the
homozygous mice. The ability of the receptor to bind DNA and
T3 was examined in gel mobility shift and
T3 binding assays, respectively, in a comparison
with the receptor from wt mice. Nuclear extracts from wt and
TR2-/- mice were incubated with and without TR- or RXR-specific
antibodies that facilitated detection of the retarded DNA bands. The
results in Fig. 3B show that the TR2 extract contains more TR1
protein than the control extract and that the overexpressed protein had
retained the ability to bind DNA. Ligand binding experiments with
125I-T3 and nuclear extract
from liver and brain of TR2-/- mice showed, as expected, a
markedly increased hormone binding as compared with extracts from wt,
TR1-/-, and TRß-/- mice (Fig. 3C). Brain nuclear extracts from
wt and homozygous animals were furthermore used in
T3 saturation binding assays to allow a better
estimation of the TR1 receptor levels. The results show that
extracts from TR2-/- mice contained about 3.7 more
T3 binding receptors than wt controls (Fig. 3D).
The data confirm that the recombination event that abrogated expression
of TR2 resulted in a concomitant increased expression of TR1 mRNA
and receptor protein.
Function of the Pituitary-Thyroid Axis
To determine the hormonal status of the mutant mice, the
concentrations of FT3 and
FT4 in serum were measured (Fig. 4A). The results show that the
FT3 and FT4 levels were
reduced in the male TR2-/- mice, and that the TR2+/- values
were intermediate between that of the wt and -/- mice. Similar
changes were detected in females. All the differences in
FT3 and FT4 were
statistically significant (see legend to Fig. 4A). Total
T3 (TT3) levels showed a
similar trend although the changes were significant only in females
(76.6 ± 2.7, 55.3 ± 2.0 and 61.0 ± 10.3 for wt, +/-,
and -/- mice, respectively). The data thus show that the genetic
alteration in the TR locus led to significantly decreased serum
levels of THs in both TR2-/+ and -/- animals.
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2+/+, +/-, and -/- mice of either sex (Fig. 4A).
We also determined pituitary expression levels for TSH and TSHß
RNAs in male mutant mice (Fig. 4B). Northern blot analysis showed no
significant differences between any of the genotypes for TSH, and a
slight increase for TSHß in TR2+/- and -/- mice (1.3- and
1.5-fold, respectively) as compared with +/+ mice. Increased levels of
TSHß but not TSH protein were also found by Western blotting of
pituitary extracts in the TR2-/- mice (Fig. 4E). However,
immunostaining histological sections of pituitaries with TSHß
antibodies revealed a normal gland morphology (Fig. 4E) and no apparent
increase in the number of TSH producing cells. In conclusion, the data
suggest that the low serum levels of THs increase TSHß RNA and
intracellular protein levels. As a corresponding increase of serum TSH
could not be demonstrated, other mechanisms must compensate to bring
serum TSH levels to normal.
Morphological and Functional Changes in Thyroid Glands
The thyroid glands of adult mice showed that the overall size of
the mutant glands was normal when compared with wt mice. In agreement
with this observation, no goiter was detected under the period of
observation (>18 months). Histological analyses of the thyroid gland
showed a general disorganization of the follicles, accompanied by a
flattening of the epithelium, as exemplified by uneven follicle sizes
and reduced interstitial space (Fig. 5A).
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2-/- mice. Serum samples for determination of
FT3 and FT4 were collected
1 wk before and 6 h after the injections. Figure 5B shows that
whereas the wt mice released serum T3 and
T4 by 48% and 75%, respectively, the levels for
TR2-/- mice increased only by 29 and 33%. These data corroborate
the histological analyses and suggest that the thyroid glands of the
mutant mice are dysfunctional.
Decreased Growth Rate and Adult Body Weight
Since TH disorders are associated with growth abnormalities, we
determined the growth rate of newborn pups as well as adult body
weight, a physiological parameter under the control of
T3. The results show that both female and male
mutant mice have a slightly reduced body weight from birth to adult
life. The growth spurt (Fig. 6A) during
the first 9 postnatal weeks, for both female and male mutant mice, was
slightly delayed when compared with wt. At 5 wk the body weight of wt
female mice was 16.9 ± 1.3 g compared with 13.5 ±
1.2 g for -/- female mice (P < 0.001). The mean
weight for 25- to 35-wk-old male wt mice was 38.1 ± 3.4 g
while TR2+/- and -/- mice weighed 34.8 ± 1.5 g and
27.6 ± 1.0 g, respectively (Fig. 6B). Similar results were
obtained for female mice of the same interval of age, with values of
34.0 ± 3.2, 31.1 ± 2.3 g, and 23.8 ± 3.0 g
for wt, TR2+/-, and TR2-/- mice, respectively. The differences
in body weight among the groups where all significant
(P < 0.05 for wt to +/- and P <
0.001 for wt or +/- to -/-). However, GH RNA and protein levels in
the pituitary were normal in TR2+/- and -/- mice (Fig. 6, C and
D). This suggests that a defect in other intermediary factors rather
than GH accounted for the growth deficiencies (see below).
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2-/- as compared with wt mice. The
TR2+/- were similar to TR2-/- mice (data not shown).
Metabolic serum parameters including serum levels of
corticosterone and leptin were not significantly altered in the
TR2-/- mice compared with wt mice (Table 2).
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shows that no differences were found in
the lengths of femurs and tibia from the wt, TR2+/-, and
TR2-/- mice. Normal longitudinal growth was further supported by
unaltered proximal tibial growth plate width in the TR2-/- mice
compared with wt (data not shown). However, serum levels of IGF-I, an
important factor involved in growth, were clearly reduced in both
TR2-/- (268 ± 28 ng/ml) and TR2+/- (308 ±17) mice
compared with wt (401 ± 22) (P < 0.05). Levels
of tartrate-resistant acid phosphatase (TRAP) 5b activity, a marker of
osteoclast activity, were reduced by 57% in the TR2-/- mice
compared with wt, whereas serum levels of osteocalcin, a marker of bone
formation, remained the same in both groups (Table 2).
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DXA measurements were performed to determine the areal bone mineral
density (BMD) and the bone mineral content (BMC). The areal BMDs and
BMCs of the femur and vertebrae L6 were
significantly reduced in the TR2-/- mice compared with wt (Table 3). In contrast, no effect was seen on
these parameters in the cranium, which consists of intramembranous bone
(Table 3). Hypothyroid rodents have a delayed mineralization of the
epiphysis. However, the epiphysis was fully mineralized in the adult
TR2-/- mice (data not shown). Furthermore, peripheral quantitative
computerized tomography (pQCT) measurements were performed to
distinguish between effects on cortical and trabecular bone. The
cortical bone was analyzed by a middiaphyseal tibial scan, which
revealed decreased cortical BMC, periosteal, and endosteal
circumferences and cortical area in both TR2+/- and -/- mice
compared with wt (Table 4). The
trabecular BMD was measured in a metaphyseal pQCT scan of the proximal
tibia and revealed a reduction with 24% in the TR2-/- mice
compared with wt, and intermediate values for the TR2+/- mice
(Table 4).
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1 have a decreased heart rate
(24), we examined whether the TR2-/- mice had heart
function alterations. Telemetric analyses show (Fig. 7) that both the TR2+/- and -/-
mice have an increased heart rate (544 ± 5 and 522 ± 18
beats/min, respectively) as compared with control mice (491 ± 6.5
beats/min). Daily administration of T3 for 4
d (as indicated by arrows) resulted in a similar and
parallel increase in heart rate (about 50 beats/min) in the three
groups, showing that the mice respond to T3.
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1
expression or the abrogation of TR2 affected the body temperature of
the mutants. Our results show that -/- (36.9 ± 0.1 C) and +/-
(36.7 ± 0.1 C) mice had an increased body temperature as compared
with wt controls (36.5 ± 0.1 C). Moreover,
T3 injection resulted in an increase of 0.5 C in
all the three genotypes. Our telemetry studies thus revealed
unexpectedly that the +/- and -/- mice exhibited features usually
associated with an hyperthyroid phenotype, despite their low levels of
FT3 and FT4. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2-Deficient Mice2 RNA, but also generated a shorter TR1 transcript expressed
at markedly higher levels than the normal TR1 RNA in all the tissues
analyzed. Because the shorter TR1 RNA resulted in a receptor with
unaffected ability to transactivate (data not shown) and to bind
T3 and DNA (Fig. 3), the mutant mice can be
considered to lack detectable TR2 expression while overproducing the
TR1 protein.
The alteration in the TR locus resulted in breeding abnormalities.
TR2-/- intercrosses rarely generated litters, a problem that was
exacerbated after the mutation was backcrossed for two generations onto
the C57BL/6J background (Forrest, D., data not shown). TR2-/-
males were fertile while females showed partial fertility but only
under optimized animal care conditions (Vennström, B.,
unpublished); thus, there is no absolute requirement for TR2 in
reproduction. TR2-/- females had a somewhat prolonged estrous
cycle, suggesting that the reduced fertility reflected irregular
ovulation. More severe estrous cycling defects have been found in mice
lacking all known T3 receptors
(TR1-/-ß-/-; Wang, Z., and D. Forrest, unpublished
data). Our results suggest that TR2 is dispensable for
reproduction and indicate that the overexpression of TR1, as
exemplified by the TR2+/- females, may be a major interfering
factor in reproduction.
Function of the Pituitary-Thyroid Axis
During normal conditions, a reduction of T3
in serum causes an increase in serum TSH. The lower
T3 and T4 levels in the
hetero- and homozygous mice, however, were not reflected by elevated
TSH levels. Thus, defects in TSH regulation cannot be the sole cause of
the low TH levels in the mutant mice. Our results suggest a double
impairment in the pituitary-thyroid axis: an inability of the thyroid
gland to produce hormone, and an alteration in the negative feedback at
the hypothalamic-pituitary level, which may also include a defect in
TRH response.
A deficiency in thyroid gland function is supported by the mild
morphological changes and the decreased response to TSH (Fig. 5). The
underlying defect is at present unclear, but could entail a subtle
misdevelopment of the gland, decreased ability to bind TSH, or defects
in the pathways of hormone synthesis or release.
The failure to elevate serum TSH in response to the slightly lowered
T3 and T4 levels indicates
another defect: in TSH regulation at the hypothalamic-pituitary level.
As may be predicted, TSHß mRNA and protein levels within the
pituitary were somewhat up-regulated (Fig. 3A). However, the lack of an
accompanying increase in serum TSH levels suggested a possible defect
in the regulation of the assembly or secretion of mature TSH by the
pituitary thyrotropes (33). Such a subtle defect could
reside within the pituitary or potentially at the level of hypothalamic
TRH. TR2 is coexpressed with the TR1 and TRß
T3 receptors in the hypothalamus and pituitary
(34), and abnormalities in TRH (35) and TSH
expression occur in the absence of TR1 and TRß (29, 36). It is also possible that the defects in regulation of
pituitary TSH result in secretion of TSH with reduced biological
activity, as was suggested for mice with a deletion of the TRH gene
(37).
The observation that TR2+/- animals had normal serum TSH and TH
levels intermediate between those of wt and -/- mice (Fig. 4)
indicates that elevation of TR1 expression impedes the function of
the pituitary-thyroid axis, although an effect of the reduction in
TR2 expression cannot be completely ruled out. That TR1 can act
in regulation of TSH synthesis is supported by comparing mice that lack
TR1, TRß, or both receptors: only the total lack of
T3 binding receptors resulted in serum TSH levels
equal to those found in severely hypothyroid wt mice (29, 30, 36).
Skeletal Properties
TR2-/- mice showed slightly reduced body weight and decreased
serum levels of IGF-I. In contrast, no significant reduction in
longitudinal bone growth was detected. However, the mice showed
decreased cortical BMC and reduced dimensions of the tibial cortical
bone. These parameters are associated with decreased adult periosteal
growth of bones and indicate late-onset growth retardation in the mice.
Interestingly, the heterozygous mice displayed a similar phenotype of
the cortical bone as the TR2-/- mice, caused by either
overexpression of TR1 or the reduction in TR2 expression, or
both. It has previously been reported that TR1-/- and TRß-/-
mice lack overt growth phenotype (24, 25). In contrast,
TR-/- mice (that express neither TR1 nor TR2 but instead
truncated versions thereof) show growth inhibition from 2 wk of age
(30, 35). Moreover, TR1-/-ß-/- mice exhibit pre-
and postnatal growth retardation as a consequence of GH/IGF-I
deficiency and exhibit compensatory growth in response to GH
substitution (Refs. 29 37A ).
The fact that TR2+/- and -/- mice had femurs and tibias of normal
length but reduced cortical dimensions suggest that the inhibition of
growth occurred after the main part of longitudinal bone growth had
taken place. The observation of growth retardation affecting the
cortical dimensions of bones but not longitudinal growth is further
strengthened by the finding that the homozygous mice had normal total
growth plate width as well as width of the individual hypertrophic and
proliferative layers. The effects of GH on bone include increased
longitudinal bone growth in young mice and periosteal bone formation in
adult mice (38). Serum bone markers, including serum
levels of osteoclast-derived TRAP 5b activity and osteoblast-derived
osteocalcin, are often used as indicators of acute changes in bone
metabolism. The BMD was decreased in the TR2-/- mice, but the bone
metabolism had probably reached a new steady state in these mice as the
osteocalcin levels were unchanged. We have previously seen that TRAP 5b
activity is well correlated to the trabecular bone mineral density when
the bone metabolism has reached a steady state (Ohlsson, C.,
unpublished). Thus, the decreased TRAP 5b activity in the TR2-/-
mice might be due to the decreased trabecular bone mineral density
associated with a decreased total number of osteoclasts.
A plausible explanation for the adult-onset growth retardation in the
mice is that either the lack of TR2 or the increased TR1
expression results in a late-onset IGF-I deficiency. That both hetero-
and homozygous mice exhibit similar alterations of the tibial cortical
bone excludes deficiency of the TR2 isoform alone as the direct
cause of these alterations. In contrast, trabecular BMD was clearly
decreased in homozygotes while only a tendency to decrease was seen in
heterozygous mice, indicating that the TR2 isoform may be of
importance for trabecular bone. Interestingly, the IGF-I reduction is
probably GH independent since no major changes in GH were detected.
T3 is required for a normal terminal
differentiation of chondrocytes (39), which is necessary
for a proper mineralization. Accordingly, bone maturation is inhibited
and there is a mineralization defect in hypothyroid rats (40, 41) as well as in TR-/- and TR1-/-ß-/- mice, but
not in TR1-/- or TRß-/- mice. Adult TR2-/- mice have low
T3 but overexpress the 1 isoform, and they
exhibit normal mineralization of the epiphysis. These results indicate
that TR2 alone is not essential for skeletal mineralization.
TH is known to affect basal metabolism of almost all cells, and
pathological conditions of the thyroid axis often affect body weight.
The increased fat content in both TR2-deficient mouse strains
contrasts with the lower overall body weight and normal cortical bone
length. The increased fat content is a feature of hypothyroidism
(42), but the lower body weight is a feature of
hyperthyroidism. This is likely to reflect distinct
physiological mechanisms governing the development of the concerned
tissues.
Heart Rate and Body Temperature
The heart is a major target for TH. Deficiency in TR isoforms
causes discrete alterations in heart rate and ventricular function
(24, 28, 43, 44). Notably, loss of TR1 in mice causes
an approximately 20% reduction in basal heart rate while still
allowing T3 to increase the heart rate. This
contrasts with the cardiac effects of TRß deficiency: a minor
increase in basal heart rate (likely to be due to slightly increased
serum levels of T3) and an impaired heart rate
response to injection of T3 into hypothyroid mice
(28, 44). The mice heterozygous and homozygous for the
TR2 ablation both exhibited a 10% increase in basal heart rate as
compared with wt controls, despite their lower serum
T3 levels. This indicates that the increase in
TR1, as opposed to loss of or reduction in TR2, may be the cause
for the elevated heart rate.
Deficiency for TR1 but not TRß results in a lowered body
temperature (24). Interestingly, both the hetero- and
homozygous TR2 mutant mice showed a similar increased temperature,
indicating that elevated TR1 expression causes this physiological
response.
Relative Contributions of TR1 Overexpression and Loss of
TR2
Recently, Macchia et al. (44) described
pituitary-thyroid axis function in TR0/0 mice, a strain that
lacks expression of all TR isoforms. These mice exhibit an increased
sensitivity to T3 in regulation of target genes,
attributed by the authors to a silencing effect of TR2. Our results
from the TR2-deficient mouse strains do not refute or support this
suggestion since the ablation of TR2 expression was accompanied by
an inevitable increase in TR1, thus making it difficult to
discriminate between a role for TR2 in modifying a hormonal response
and an increased activity of TR1.
Because of the reduced serum levels of TH one could expect that the
hetero- and homozygous TR2 mice would show a hypothyroid phenotype
in target tissues. However some of the alterations observed, including
decreased body weight, increased heart rate, and increased body
temperature, are consistent with a hyperthyroid phenotype. It is
possible that the increase in TR1 could lead to an increased
activity of the receptor, provided intracellular availability to
hormone is adequate.
The observation that the heterozygous mice in all aspects studied in
this report exhibited a phenotype intermediate or identical to that of
homozygotes suggests that the loss of TR2 has had a much smaller
impact as compared with the increased expression of TR1. The
phenotypic changes described by us cannot be attributed solely to the
loss of TR2; in fact, they could with few exceptions be explained as
an effect of overexpressed TR1. The effects of TR2 protein on
T3-mediated gene regulation in vitro
have previously been described to be weak (18), suggesting
that further analyses of the finer details of TH action in our mouse
strain is required to assess the role of TR2 in physiology.
The discovery of TR2 in mammalian, but not amphibian, avian, or fish
species despite extensive investigation, suggests that a role for
TR2 would represent only a refinement of the basic function of the
(Thra) TR gene. Accordingly, TR2 may be viewed as having an
active role in control of normal TR signaling, or at another extreme,
as having no physiological role but instead representing a consequence
of how the mammalian genome arose. The phenotypes led us to consider
the explanation that the ability to divert TR gene expression toward
production of TR2, irrespective of if it has an activity, may
represent a means for adjusting the activity of the
T3 binding TR1 protein to physiologically
appropriate levels. Our data neither refute nor support these
alternative hypotheses but do allow us to conclude that the ratio of
expression of TR1:TR2 plays an important, widespread regulatory
role in mammalian physiology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gene contains 10 exons (45), which give
rise to two different mRNAs. The first nine exons encode the TR1
protein, whereas the TR2 mRNA is generated from alternative splicing
from a donor site 128 bp after the start of exon 9 to the acceptor site
for exon 10. Because little is known about the individual role of the
TR1 and TR2 proteins, we decided to delete independently the
TR1 (24) and the TR2 genes. To generate the TR2
mutant mice, we modified our TR1 targeting vector (24),
so that it contains the entire coding sequence of exon 9, followed by a
strong SV40 polyadenylation signal and a thymidine kinase (TK)
promoter-neo cassette pMCneo/poly(A) (Stratagene, La
Jolla, CA) in the opposite transcriptional orientation. The strong SV40
polyadenylation site blocked transcription into exon 10, effectively
abrogating expression of the TR2 protein. Exon 10 of the TR gene
and the last exon of the rev-erbA gene were left intact, thus
avoiding disturbances of transcription of rev-erbA.
Generation of TR2 Mutant Mice
Homologous recombination in E14 embryonic stem cells (ES cells)
and the screening of positive clones were done as previously described
(24). The same probes were used in the Southern blot
analyses: 5'-probe (5'p) recognizing a 23-kb fragment from the wt
allele and a 13-kb band from the mutated allele; the 3'-probe (3'p)
detecting the 23-kb band from the wt allele and a 9.9-kb band from the
mutated allele (see Fig. 1B). An exon 10 probe (ex10p) was used as an
internal probe to exclude the presence of extra bands due to complete
or partial integration of the targeting vector into the putative
positive clones. Two more independent DNA digestions (XbaI
and StuI) (Fig. 1B) were carried out to confirm the correct
integration of the targeting event. The ex8–9 probe, PCR generated
from genomic DNA with a 5'-primer at the end of exon 8 (Ex8.5') and a
3'-primer at the start of exon 9 (Ex9.3') detected the expected bands,
a 2.8-kb and a 5.6-kb bands from the wt and the mutated allele,
respectively, in the XbaI digestion; and a 1.3-kb band from
both alleles after StuI digestion.
Blastocyst injections with the positive clone 165 were performed as described previously (24). From the chimeras generated, one male transmitted the mutation to its offspring when crossed with BALB/c female, yielding mice with a mixed 129/Ola x BALB/c genetic background. Due to poor reproduction and pup rearing by females carrying the targeted allele, +/- and -/- mice were generated by female +/- x male -/- intercrosses, whereas wt animals were generated by +/+ x +/- or +/+ crosses. The wt and mutant lines were intercrossed every two to three generations to avoid genetic drift between them. Some experiments were carried out with wt and mutant mice derived from crossing the 129/Ola+BALB/c line with mice having a mixed 129/sv and C67/B6J background. Mutant and wt mice from this line of mice were generated as described above. This line had an improved, but still impaired, reproduction. They differed little from the original strain in other respects. Pups were genotyped at 2–3 wk of age.
The animals were kept at the facilities in New York and Stockholm as
described previously (29). The specifics for breeding the
TR2 mice can be obtained from B. Vennström upon request.
For readability, we will denote the mouse strains described above as
TR2-/-. However, the mice will be deposited at The Jackson Laboratories (Bar Harbor, ME) under the name
Thratm2Ven/tm2Ven. The mouse strains
TR1-/- and TRß-/- are available at The Jackson Laboratories under the designations
Thratm1Ven and
Thrbtm1Df.
Estrous Cycle
Estrous cycling was determined by examination of vaginal smears
taken daily from mice that were housed individually. Smears were taken
at approximately the same time every day for up to 40 d.
RT-PCR
Heart, liver, and brain were dissected from adult mice (3 months
old). Heterozygous and homozygous embryos were taken at E15 and wt
embryos at E13. RNA was prepared with the Ultraspec kit (Biotex
Laboratories, Inc., Houston, TX) according to the supplier’s
instructions. Total embryo or 100 mg of tissue were used for RNA
preparation. cDNA was obtained with the SuperScript preamplification
system (Life Technologies, Inc., Gaithersburg, MD) using an oligo-dT
primer. The amount of RNA used for each cDNA preparation varied from
1.1 to 4 µg. To perform each PCR reaction we used one-tenth of the
cDNA or 110–400 ng of total RNA. The primers used in the PCR reaction
were: 5'-Sal ex9 hybridizing to the beginning of exon 9 of the TR
locus; UTex9, 3'-primer recognizing the untranslated region of exon 9,
only present in the wt allele; SV40, 3'-primer recognizing a specific
sequence of the SV40 polyA+, introduced and present only in the mutated
allele; 5'-ex8-1, 5'-primer recognizing exon 8; 3'-ex9-1, 3'-primer
hybridizing to exon 9; 5'-ex8-2, 5'-primer recognizing exon 8 and
finally 3' ex10-2, 3'-primer hybridizing to exon 10. PCRs were
performed according to the SuperScript preamplification system
recommendations, with 57 C annealing temperature for 35 cycles.
PCR Primer Sequences
5'-Sal ex9: 5'-GGA GTC GAC CGA GAA GAG TCA GGA UTex9: 5'-CAG
GGG AAA TCT AGG CCA AGG AAC SV40: 5'-CAC TGC ATT CTA GTT GTG GT
5'-ex8-1: 5'-TCC GCT ACG ACC CTG AGA GTG AC 5'-ex8-2: 5'-GAA TGG
TGG CTT GGG TGT GGT CT 3'-ex9-1: 5'-TGG GAG GAA GGA GAG AAG AGA
T 3'-ex10-2: 5'-GAC CTG CGG ACC CTG AAC AAC Ex8.5': 5'-GGC TGT
GCT GCT AAT GTC AAC Ex9.3': 5'-GCG TCG ACA GCA AGT TCATTT ATG
GCC
Northern Blot Analysis
Polyadenylated RNAs were prepared from tissues as previously
described (46). Northern blots were performed as before
(47), and the probes used for the analyses were the
complete cDNA of TR1 recognizing both TR1 (5.8 kb) and TR2
(2.6 kb) variants, specific probes for the two isoforms TR1 and
TR2, and cDNA probes for TSH and TSHß. Levels of
expression were normalized to the expression of
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA.
Quantification was done with PhosphorImagers from Fuji Photo Film Co., Ltd. (Stamford, CT) or Molecular Dynamics, Inc. (Sunnyvale, CA).
Gel Mobility Shift Assays and Nuclear T3 Binding
Determinations
Nuclear extracts from brain and liver of wt and homozygous mice
were prepared and analyzed using an F2 TRE as
earlier described (29). Ligand binding experiments were
performed as previously described (29) to determine the
extent to which T3 binding was increased because
of TR1 overexpression.
Hormone Assays
FT3 and FT4 were
obtained from 1.5 to 5-month-old mice using a competitive RIA
kit (Amerlex-MAB FT3 and FT4 kit) from Amersham Pharmacia Biotech (Piscataway, NJ) as described before (24).
TSH and total T3 and total
T4 levels in serum were determined as previously
described (48). Recombinant bovine TSH was purchased from
Sigma (St. Louis, MO). GH levels were measured as
described earlier (49). The computer program StatView 4.5
(SAS Institute Inc., Cary, NC) was used in ANOVA analyses to
compare all the groups in our hormone assays and also to compare weight
and growth curves.
Histology and Immunostaining
Thyroid glands of wt and TR2-/- mice were embedded in
paraffin, sectioned (5 µm thick), and stained with hemotoxylin and
eosin according to standard protocols. TSH immunostaining was performed
as previously described (29).
Serum Parameters
Serum IGF-I levels were measured by double-antibody IGF binding
protein-blocked RIA (49).
Serum osteocalcin levels were measured using a monoclonal antibody raised against human osteocalcin (Rat-MID osteocalcin ELISA, Osteometer Biotech, Copenhagen, Denmark). The sensitivity of the osteocalcin assay was 21.1 ng/ml, and intra- and interassay coefficients of variation (CVs) were less than 10%. Serum leptin levels were measured by a RIA (Crystal Chem, Inc., Chicago, IL) with intra- and interassay CVs of 5.4 and 6.9%, respectively. Serum corticosterone levels were measured by a RIA (ImmunoChem, ICN Biomedicals, Inc., Costa Mesa, CA) with intra- and interassay CVs of 6.5 and 4.4%, respectively.
Measurement of TRAP activity was performed with the TRAP 5b immunoassay (50). TRAP was purified from human osteoclasts as described, and the purified enzyme was used as antigen to develop a polyclonal TRAP-antiserum in rabbits. A 1:1,000 dilution of the antiserum was used. In the immunoassay, the antiserum was incubated on antirabbit IgG-coated microtiter plates (EG & G Wallac, Inc., Turku, Finland) for 1 h. Diluted mouse serum samples (200 µl) were incubated in the wells for 1 h, and bound enzyme activity was detected using 8 mmol/liter 4-nitrophenyl phosphate as substrate in 0.1 mol/liter sodium acetate buffer, pH 6.1, for 2 h at 37 C. The enzyme reactions were terminated by adding 25 µl of 0.32 mol/liter NaOH to the wells, and A405 was measured using model 2 Victor equipment (EG & G Wallac, Inc.).
DXA
BMC and areal BMD (BMC/cm2) were measured
as described earlier (29) using the pDEXA Sabre
(Norland Medical Systems, Inc., Fort Atkinson, WI) with
Sabre Research 3.6 software. In vivo measurements were
performed with three mice in the same scan. To avoid interscan
variations a wt mouse was included as an internal control in each scan.
Ex vivo measurements of the left femur and tibia and
vertebrae L6 were performed on excised bones
placed on a 1-cm-thick Plexiglas table. High-resolution scans were
performed (line spacing: tibia and femur, 0.02 cm; vertebrae, 0.01 cm).
In vivo measurements of animals were performed to measure
total body BMC. Medium resolution scans were performed (line spacing,
0.05 cm).
Peripheral Quantitative Computerized Tomography
Tomographic measurements were performed using the STRATEC pQCT
XCT (software version 5.4B; Norland Medical Systems, Inc.)
operating at a resolution of 70 µm as previously described
(51).
Histological Staining and Growth Plate Measurements
Right femurs were excised and fixed in 4% buffered
paraformaldehyde, and were subsequently decalcified, embedded in
paraffin, and sectioned. Sections were stained with Alcian Blue/van
Gieson stain. The width of growth plates was measured as previously
described (30). For measurements of total growth plate and
the hypertrophic layer, the average of 30 measurements was calculated.
The width of the proliferative layer was calculated by subtracting the
width of the hypertrophic layer from the width of the total growth
plate.
Fat Measurements
We have previously developed a combined DXA image analysis
procedure for the in vivo prediction of fat content in mice
(52). The interassay CV for the measurements of percent
fat area was less than 3%.
Telemetry
Telemetry assays were performed in adult mice (3–5 months old):
seven wt, seven heterozygous, and six homozygous mice.
Electrocardiogram records, body temperature, and locomotor activity
were analyzed as described previously (53). The mice were
allowed to recover at least 7 d before the recordings of each
individual mouse kept in its own cage were begun. After 48 h of
baseline registration, the animals were injected daily with
T3 (Sigma, St. Louis, MO; 0.1 mg/kg
sc) for 4 d at 1300 h.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Laboratory of Molecular Neurobiology, Department of
Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77
Solna, Sweden. 
Abbreviations: BMC, Bone mineral content; BMD, bone mineral density; CV, coefficient of variation; DXA, dual x-ray absorptiometry; G3PDH, glyceraldehydes-3-phosphate dehydrogenase; pQCT, peripheral quantitative computed tomography; TH, thyroid hormone; TRAP, tartrate-resistant acid phosphatase; TRE, thyroid hormone response element; wt, wild type.
Received for publication June 21, 2001. Accepted for publication August 27, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), TR
), TR
). Mean values
are indicated by bars. All the differences are
significant, P < 0.001, except for FT3
in females +/- vs. -/- and for FT4 in
males and females +/- compared with +/+ or -/- mice, where
P < 0.05. B, TSH levels in serum from control,
TR
) TR
), and wt (