Download The Selective Loss of the Type 2 Iodothyronine Deiodinase in

T H Y R O I D - T R H - T S H
The Selective Loss of the Type 2 Iodothyronine
Deiodinase in Mouse Thyrotrophs Increases Basal TSH
but Blunts the Thyrotropin Response to
Hypothyroidism
Cristina Luongo, Cecilia Martin, Kristen Vella, Alessandro Marsili,
Raffaele Ambrosio, Monica Dentice, John W. Harney, Domenico Salvatore,
Ann Marie Zavacki, and P. Reed Larsen
Thyroid Section, Division of Endocrinology, Diabetes, and Hypertension, Department of Medicine (C.L.,
C.M., A.M., J.W.H., A.M.Z., P.R.L.), Brigham and Women’s Hospital and Harvard Medical School, and
Division of Endocrinology, Diabetes, and Metabolism (K.V.), Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, Massachusetts 02115; Istituto di Ricovero e Cura a Carattere Scientifico
Fondazione Studio di Diagnostica Nucleare “SDN” (R.A.), 80142 Naples, Italy; and Department of
Clinical Medicine and Surgery (M.D., D.S.), University of Naples Federico II, 80131 Naples, Italy
The type 2 iodothyronine deiodinase (D2) is essential for feedback regulation of TSH by T4. We
genetically inactivated in vivo D2 in thyrotrophs using a mouse model of Cga-driven cre recombinase. Pituitary D2 activity was reduced 90% in the Cga-cre D2 knockout (KO) mice compared with
control Dio2fl/fl mice. There was no growth or reproductive phenotype. Basal TSH levels were
increased 1.5- to 1.8-fold, but serum T4 and T3 were not different from the controls in adult mice.
In hypothyroid adult mice, suppression of TSH by T4, but not T3, was impaired. Despite mild basal
TSH elevation, the TSH increase in response to hypothyroidism was 4-fold reduced in the Cga-cre
D2KO compared with control mice despite an identical level of pituitary TSH ␣- and ␤-subunit
mRNAs. In neonatal Cga-cre D2KO mice, TSH was also 2-fold higher than in the controls, but serum
T4 was elevated. Despite a constant TSH, serum T4 increased 2–3-fold between postnatal day (P) 5
and P15 in both genotypes. The pituitary, but not cerebrocortical, D2 activity was markedly elevated in P5 mice decreasing towards adult levels by P17. In conclusion, a congenital severe reduction of thyrotroph D2 causes a major impairment of the TSH response to hypothyroidism. This
would be deleterious to the compensatory adaptation of the thyroid gland to iodine deficiency.
(Endocrinology 156: 745–754, 2015)
A
n important role for serum T4 in the feedback regulation of TSH secretion was first suspected from the
presence of an elevated serum TSH in combination with a
normal serum T3 and a reduced serum T4 in moderately
hypothyroid patients (1). This was initially confusing, because in many bioassay experiments, T3 appeared to be the
active hormone, and it was already recognized that T4 to
T3 conversion occurred (2). Subsequent studies demonstrated that in the hypothyroid rat, the acute suppression
of TSH secretion by T4 did not require the formation of
circulating T3 but did depend on T3 produced from T4
5⬘-deiodination within the pituitary by an unidentified,
propylthiouracil (PTU)-resistant, 5⬘-deiodinase (3, 4).
Both the intrapituitary T3 production and the suppression
of TSH in hypothyroid rats were blocked in parallel by the
iodophenol, iopanoic acid, a competitive inhibitor of deiodination (4, 5). Further studies showed that this PTUresistant deiodinase, subsequently termed type 2 iodothyronine deiodinase (D2), was present in pituitary thyrotrophs,
mammotrophs, and somatotrophs as well in the central ner-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2015 by the Endocrine Society
Received August 19, 2014. Accepted November 25, 2014.
First Published Online December 2, 2014
Abbreviations: Cga, glycoprotein hormone ␣-subunit gene; CNS, central nervous system;
D2, type 2 iodothyronine deiodinase; DHA, dorsal hypothalamic area; KO, knockout; MBH,
mediobasal hypothalamus; MMI, methimazole; P, postnatal day; PTU, propylthiouracil;
PVN, paraventricular nucleus; TR, thyroid hormone receptor.
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Pituitary Dio2 Role in Hypothyroidism
vous system (CNS) (largely in astrocytes) and in brown adipose tissue (6).
Interestingly, the activity of D2 in pituitary cells was
decreased by thyroid hormone, whereas that of the hepatic
D1 was induced at a transcriptional level by the same
stimulus (7). Subsequent molecular cloning of both D1
and D2 confirmed that although these proteins were both
selenoenzymes that were highly homologous especially in
their active centers, they are markedly different in tissue
distribution and function and are encoded by different
genes (8, 9). The identification of pituitary D2 provided a
mechanism through which the thyrotroph could recognize
and respond to a decrease in the circulating prohormone
T4. Although a global D2 knockout mouse has been developed and shows a clear defect in T4 responsiveness in
the pituitary-thyroid axis, the absence of D2 expression in
nonpituitary tissues, such as the CNS, complicates the interpretation of the role of thyrotroph D2 deficiency, per se
(10, 11).
To define the effects of a selective thyrotroph D2 deficiency, we crossed a mouse with a floxed Dio2 gene with
one in which cre-expression is under control of the glycoprotein hormone ␣-subunit gene (Cga), thus creating a
model in which active D2 was depleted in the Cga-expressing thyrotrophs and gonadotrophs, the Cga-cre D2
knockout (KO) mouse (12). This allowed us to examine
the functional role of thyrotroph D2 in the euthyroid and
hypothyroid state. We found that Cga-cre D2KO mice
have an approximately 90% loss of D2 in the pituitary, a
normal serum T3 and T4, and a slightly higher but still
normal TSH, whereas TSH bioactivity is unchanged.
Strikingly, we also found that Cga-cre D2KO mice have an
impaired TSH increase during induction of the hypothyroid state. While this work was in progress, Fonseca et al
(13) reported studies of adult male mice with inactivated
pituitary D2 using the same strategy. Unlike our results,
they reported an increase in serum T4 and a goiter despite
a normal circulating TSH. A detailed comparison of other
differences in the results of the 2 studies is provided in the
Discussion. In terms of selective environmental pressures,
dietary iodine deficiency is the most serious challenge that
the hypothalamic-pituitary-thyroid axis faces in maintaining T3 homeostasis (14). Our results indicate that thyrotroph D2 plays a critical role in meeting that challenge.
Materials and Methods
Animals
All animal studies were approved by the Harvard Medical
School Standing Committee on Animals. Mice were fed standard
rodent chow and water ad libitum and maintained under a 12-
Endocrinology, February 2015, 156(2):745–754
hour light, 12-hour dark cycle. For experiments with neonates,
whole litters were euthanized at the indicated time point by isoflurane anesthesia followed by decapitation, and serum and tissues were collected. Mice were then genotyped, and sufficient
litters killed to allow for at least 3 mice (range 3–7) for each
genotype and gender at each time point. The same animals were
used for measurement of serum hormone levels (see Figure 5
below) and tissue deiodinase measurements (see Figure 6 below).
Generation of D2fl/fl mice
Initially, a 9132-bp fragment containing exon 2 of the mouse
Dio2 gene cloned from BAC: bMQ163j10 (Source BioScience)
was inserted by recombination into the PL253 plasmid (15). A
LoxP site and a neomycin expression cassette were inserted into
the single intron, whereas another LoxP site was inserted into the
3⬘ untranslated region in exon 2 (Supplemental Figure 1). This
construct was confirmed by restriction digests and sequencing
and electroporated into mouse ES cells (E14Tg2A). Geneticinresistant clones were analyzed by PCR and Southern blotting and
used for blastocyst injection by the BWH transgenic core. Mice
bearing the floxed allele of the Dio2 gene were bred to homozygosity and are referred to as D2fl/fl mice. D2fl/fl mice were crossed
with transgenic mice obtained from The Jackson Laboratory
(B6;SJL-Tg[Cga-Cre]3Sac/J) developed by Dr Sally Camper (12),
in which Cre expression was under control of the mouse Cga.
This specifically deleted the floxed fragment of the Dio2 gene in
the Cga-expressing thyrotrophs and gonadotrophs of the anterior pituitary. Earlier studies showed that there was low and
scattered expression of this promoter in the brain and that Cgacre expression did not affect TSH expression in thyrotrophs
(12, 16).
Serum T3, T4, and TSH measurements
Blood was either collected from the cheek vein, or by terminal
cardiac puncture under isoflurane anesthesia, and serum T3 and
T4 were measured using COAT-A-COUNT total T4 and T3 kit
(DPC), following the manufacturer’s instructions. The mouse
standard curves were prepared in charcoal-stripped (T4 and T3
deficient) mouse serum as described previously (17). 125I-T3
charcoal uptake was assessed as described previously (18). Serum TSH levels were measured by Milliplex MAP using the rat
thyroid hormone TSH panel (EMD Millipore) (19).
Induction of hypothyroidism and T4 sensitivity
testing
To induce hypothyroidism, Cga-cre D2KO and D2fl/fl control mice were administered 0.1% methimazole (MMI) (Sigma)
and 1% NaClO4 (Fisher Scientific Co) in drinking water for 3– 6
weeks as indicated. The sensitivity of the elevated TSH to acute
suppression by T4 or T3 was performed as described previously
(11). In brief, mice were made hypothyroid by administration of
1% NaClO4 and 0.1% MMI in their drinking water for 3 weeks.
On the day of the experiment, blood was obtained from the cheek
vein for determination of basal TSH levels. Mice were then injected ip with PTU (2 mg/mouse) to block D1 activity, and 1 hour
later given either T4 (3 ␮g/100 g body weight) or T3 (1.2 ␮g/100
g body weight) or vehicle alone (PBS). Five hours after hormone
administration, mice were euthanized by cardiac puncture under
isoflurane anesthesia, and serum and tissues were collected.
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Deiodination assays
D1, D2, and D3 activities were assayed as described previously (18).
Gene expression analysis
The hypothalamic tissue was collected using the coronal brain
matrix (Braintree Scientific). The hypothalamic region was dissected by coronal cuts located caudal to the optic chiasm and
rostral to the mammillary bodies followed by 2 lateral cuts 1 mm
to either side of the midline and 2.2 mm from the dura (20). We
separated this tissue into 2 regions performing an additional
dorso-ventral cut 1 mm from the dura: 1) the upper hypothalamus containing the dorsal hypothalamic area (DHA) and the
paraventricular nucleus (PVN) and lateral hypothalamic tissue,
and 2) the lower hypothalamus containing the mediobasal hypothalamus (MBH), including tanycytes, the arcuate nucleus,
and the median eminence. Total RNA was extracted from tissues
using TRIzol (Invitrogen), according to the manufacturer’s di-
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rections. One microgram of total RNA was reverse transcribed into
cDNA using SuperScript VILO (Invitrogen). The cDNAs were amplified by PCR in an i-Cycler (Bio-Rad) using SYBR Green (BioRad). Sequences of primers used are listed in Supplemental Table 1.
Quantitative real-time PCR was performed as described previously
using the delta delta cycle threshold method, with cyclophilin A
used as the housekeeping gene for normalization (21).
TSH biological activity
TSH bioactivity was measured as described previously, with
JP26 –26 and JP02 cells generously provided by Dr G. Vassart and
Dr S. Refetoff (17, 22, 23). cAMP was measured using either the
cAMP [125I] RIA kit, catalog number NEK033 (PerkinElmer) for
sera from euthyroid mice, or cAMP XP kit for sera from hypothyroid mice (Cell Signaling Technology). The biological activity
of TSH in each serum sample was determined by dividing the net
pg cAMP induced/pg TSH.
Results
Figure 1. D2 Activity in control and Cga-cre D2KO mice. D2 activity in pituitary (A) and cerebral
cortex (B) of euthyroid (left) and hypothyroid (right) animals. C, D2 activity in hypothalamic areas
under euthyroid conditions: DHA (left) and MBH (right). Control animals are shown in the white
bars and the Cga-cre D2KO animals with black bars. Data are shown as mean ⫾ SEM. *, P ⬍
.05; **, P ⬍ .01; ***, P ⬍ .001. n ⫽ 3–5 mice per group.
Phenotype and pituitary,
cerebrocortical, and
hypothalamic deiodinase
activities in Cga-cre D2KO mice
We generated mice with 2 LoxP
sites flanking the Dio2 gene (Supplemental Figure 1) that were mated
with mice harboring a cre recombinase expression cassette under the
control of the mouse Cga to remove
a portion of the Dio2 coding region
from the Cga-expressing thyrotrophs in the pituitary (hereafter
Cga-cre D2KOs) (12). The Cga-cre
D2KO mice had no significant differences in body weight, growth, litter size, or reproductive capacity in
either gender when compared with
D2fl/fl littermates (data not shown).
These findings are consistent with
similar growth hormone mRNA levels in euthyroid 8-week-old Cga-cre,
D2KO mice (n ⫽ 6) when compared
with age-matched controls (n ⫽ 4,
P ⫽ .6723) (data not shown).
D2 activity was quantified in several tissues to confirm successful and
specific recombination. Pituitary D2
was reduced approximately 90% in
the euthyroid Cga-cre D2KO mice
relative to that in controls indicating
significant recombination of the
Dio2 gene (P ⬍ .01) (Figure 1A). Al-
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Pituitary Dio2 Role in Hypothyroidism
though the D2 activities in both genotypes were about 10fold higher when mice were made hypothyroid for 4 – 6
weeks, D2 activity was still 90% lower in the Cga-cre D2KO
mice compared with the controls (P ⬍ .001) (Figure 1A). The
residual activity in the pituitary of Cga-cre D2KO mice can
be explained by either by incomplete recombination and/or
the previously demonstrated D2 activity in somatotrophs
and mammotrophs (24, 25). D2 activity was reduced 25% in
the cerebral cortex of euthyroid Cga-cre D2KOs compared
with controls (P ⬍ .05), but this difference in D2 activity was
lost in hypothyroid mice (Figure 1B). In addition, D2 activity
in the DHA containing the PVNs and in the MBH, which
included the D2 expressing-tanycytes, was similar between
the 2 genotypes (Figure 1C). Likewise, D3 activities were
unchanged in pituitary, cerebral cortex, or DHA and MBH
of Cga-cre D2KO and control mice (Supplemental Figure 2).
We also found no thyroid enlargement despite the slightly
higher TSH in the male Cga-cre, D2KO mice (data not
shown).
The serum TSH concentrations in the adult male and female Cga-cre D2KO mice were 1.5- to 1.8-fold higher, with
this difference not reaching statistical significance in the females (Table 1). Despite the slightly higher TSH, the serum
T4 and T3 concentrations in control and Cga-cre D2KO mice
were not different. The free fraction of T3 in the Cga-cre
D2KO mice, as estimated by a T3 charcoal uptake determination, was not different from that of the control mice, indicating that differences in circulating thyroid hormone
binding proteins are not a confounding factor and that the
total hormone values are representative of the free thyroid
hormones (data not shown). We also found no differences in
the TSH bioactivity in adult (2 mo old) euthyroid male mice
of both genotypes (Supplemental Figure 3). In agreement
with the lack of a difference in thyroid hormone levels, hepatic D1 activity, a sensitive indicator of peripheral thyroid
status in rodents, was not different between the 2 genotypes
(Supplemental Figure 4).
Table 1. Basal Thyroid Function Test of Control and
Cga-cre D2KO Mice
Male (n ⴝ 22–24)
Control
Cga-cre D2KO
TSH (pg/mL)
T4 (␮g/dL)
T3 (ng/mL)
236 ⫾ 30
5.6 ⫾ 0.4
0.45 ⫾ 0.07
356 ⫾ 43a
5.5 ⫾ 0.8
0.38 ⫾ 0.06
Female (n ⴝ 6 –12)
Control
Cga-cre D2KO
TSH (pg/mL)
T4 (␮g/dL)
T3 (ng/mL)
104 ⫾ 15
5.6 ⫾ 0.5
0.42 ⫾ 0.09
186 ⫾ 38
5.8 ⫾ 0.8
0.50 ⫾ 0.04
Data as mean ⫾ SEM.
a
P ⬍ .05.
Endocrinology, February 2015, 156(2):745–754
To elucidate the effect of inactivating the pituitary Dio2
gene on the other elements of the hypothalamic-pituitarythyroid axis, we measured the pre-proTRH mRNA expression in the PVN (Supplemental Figure 5A) and pyroglutamyl peptidase II mRNA, a TRH-inactivating enzyme
expressed in tanycytes, in the MBH (Supplemental Figure
5B) by real-time PCR. The expression of these mRNAs
was unaffected in the Cga-cre D2KO mice.
Hypothyroid Cga-cre D2KO mice have impaired
TSH suppression after acute exposure to T4 but
not T3
Previous studies in hypothyroid rats showed that iopanoic acid, but not PTU, blocked the acute effect of T4,
but not T3, to decrease the elevated TSH in hypothyroid
rats. Because blockade of the TSH suppression correlated
with the inhibition of pituitary T4 to T3 conversion, this is
a D2- and T3-dependent process (4, 5). If this conclusion
is accurate, a similar phenomenon should occur in the
Cga-cre D2KO mice. To test this, control and Cga-cre
D2KO mice were made hypothyroid by the addition of
sodium perchlorate and MMI in their drinking water for
3 weeks. Mice were then injected with PTU to block D1
activity and then 1 hour later injected with either T3 or T4
(Figure 2A). Five hours after hormone injection, serum
was collected for TSH measurement. TSH was unchanged
in both genotypes after vehicle injection (Figure 2, B and
C). T4 administration caused a roughly 50% decrease in
TSH levels in the controls (P ⬍ .05) but not in the Cga-cre
D2KO animals (Figure 2, D, E, and G). On the other hand,
administration of T3 reduced serum TSH 90% in the Cgacre D2KO mice (P ⬍ .001) (Figure 2, F and G). These
results are comparable with those observed in mice with a
global loss of D2 (D2KO mice) (10, 11).
The TSH response to hypothyroidism is markedly
reduced in Cga-cre D2KO mice
To examine the effect of thyrotroph D2 deficiency on
the TSH response to antithyroid drug-induced hypothyroidism, groups of 4 male mice of each genotype were
treated with sodium perchlorate and MMI in drinking
water for 4 – 6 weeks. Serum T4 decreased to undetectable
levels over the first 2 weeks, whereas the serum T3 concentrations fell to about 1/3 of normal (Figure 3, B and C).
Strikingly, the rate of increase in TSH was markedly
slower in the Cga-cre D2KO mice than in controls, although both increased substantially (Figure 3A). At 6
weeks, the TSH concentration in the controls was approximately 3-fold greater than in the Cga-cre D2KO mice
(P ⬍ .0001). In an identical experiment in which mice were
treated with antithyroid agents for 4 weeks, serum TSH
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Serum hormone concentrations
and deiodinase activities in
neonatal Cga-cre D2KO pups
Rats exposed to excess thyroid
hormone in utero and mice born to
mothers with elevations in thyroid
hormone during gestation due to a
congenital absence of the thyroid
hormone-inactivating D3 develop
central hypothyroidism after birth
(26, 27). Because serum T4 concentrations are elevated in mice with a
global D2KO, we wondered
whether a mild neonatal thyrotoxicosis could explain the impaired
TSH response to hypothyroidism in
the Cga-cre D2KO mice. We examined the serum TSH, T4, and T3 concentrations in groups of neonatal
control and Cga-cre D2KO mice at
several times during postnatal development (Figure 5). As in the adults,
the serum TSH levels trended higher
in both male and female Cga-cre
D2KO mice at postnatal day (P) 5,
P10, and P15, although again, these
differences were generally not statistically significant (Figure 5, A and B).
A higher TSH value as a compensation
for a lower biological activity of the
Figure 2. Test of T4 sensitivity in hypothyroid control and Cga-cre D2KO mice. A, Schematic
TSH in the Cga-cre D2KO mice might
description of experiment. Mice were treated with 1% sodium perchlorate (NaClO4) and 0.1%
also explain this, but we found no difMMI in the drinking water for 3 weeks to induce hypothyroidism. The serum TSH levels were
measured in Cga-cre D2KO and control mice at time 0 (T0), (1 h after PTU pretreatment) and
ferences in this variable between genothen at T1 5 hours after ip injection of vehicle, or thyroid hormones (THs). Data from individual
types or gender (Supplemental Figure
mice are shown as the ratio T1/T0 (as %) of serum TSH values after administration of PBS (B and
3). On the other hand, unlike in the
C); 3-␮g T4/100 g body weight (D and E), or 1.2-␮g T3/100 g body weight (F) (11). Average fold
change with treatment is shown (mean ⫾ SEM) (G). *, P ⬍ .05 when compared with vehicleadult, the serum T4 concentrations are
treated control by Student’s unpaired t test, whereas ***, P ⬍ .001 compared with vehiclehigher in the Cga-cre D2KO pups, on
treated Cga-cre D2KO by one-way ANOVA. N.S., not significant. n ⫽ 4 mice/group.
days P5 and P15 in males and P5, P10,
and P15 in females (Figure 5, C and D).
concentrations in control mice were 5-fold higher (fold These, and the serum T3 concentrations, increased between
TSH increase, 1068 ⫾ 198) than in Cga-cre D2KO mice P5 and P15 but fell to normal adult levels at P17 (Figure 5, E
(fold TSH increase, 199 ⫾ 52; P ⬍ .001). These results and F). The higher TSH found in the male, as opposed to the
indicate that thyrotroph D2 is required for a normal TSH female, Cga-cre D2KO pups at P17 is consistent with higher
response to hypothyroidism.
TSH values in male adult mice (Table 1). The increases in T4
We assessed whether differences in pituitary expression values between P5 and P15 suggest a TSH-driven increase in
of the TSH ␣- and ␤-subunit mRNAs could account for the both neonatal genotypes. We found no differences in the free
decreased response of serum TSH in the hypothyroid Cga- fraction of T3 in P15 and P17 neonates between genotypes or
cre D2KO mice. The expression of the ␣ and ␤ TSH sub- gender (data not shown). Although the elevated serum T4
unit mRNAs in the euthyroid pituitary was not different in results suggested the possibility of a neonatal thyrotoxicosis,
the 2 genotypes, and there were increases of similar mag- TSH suppression did not occur.
nitude of subunit mRNAs during the induction of hypoWe also measured D2 activity in pituitary and cerebral
thyroidism (Figure 4).
cortex as well as hepatic D1 activity during the neonatal
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Endocrinology, February 2015, 156(2):745–754
males or females (Supplemental Figure 6). These results
and the similar hepatic D1 activities in both genotypes
indicate that the Cga-cre D2KO neonates are not thyrotoxic despite the higher serum T4 than in control pups.
Discussion
Figure 3. Serum TSH, T4 and T3 concentrations in control and Cga-cre
D2KO mice during induction of hypothyroidism. Mice received 1%
NaClO4 and 0.1% MMI in drinking water. Serial samples of blood were
taken for TSH (A), T4 (B), and T3 (C) measurements at the indicated
times. TSH is expressed as fold change relative to the value at time 0.
Data are shown as mean ⫾ SEM. **, P ⬍ .01; ****, P ⬍ .0001. n ⫽
3–5 mice/group.
period in these groups of Cga-cre D2KO and control neonatal mice (Figure 6). Surprisingly, the pituitary D2 in the
control male and female neonates is quite elevated at P5
(25–50 fmol T4/mg 䡠 min) compared with that at P17
(5–10 fmol T4/mg 䡠 min), or in the adult animal (3 fmol
T4/mg䡠min) (Figures 1 and 6). In males, the cerebral cortical D2 activity, a protein posttranslationally reduced by
T4, was not different between control and Cga-cre D2KO
mice despite the higher levels of T4 in the Cga-cre D2KO
mice (Figure 5, C and D). On the other hand, although not
statistically significant, cerebrocortical D2 activity
trended lower in the females at P5 and P10, but not later,
perhaps reflecting the consistently relatively greater serum
T4 in Cga-cre D2KO females (Figure 5D). Hepatic D1
activity was similar in both genotypes in males and females
(Figure 6, E and F). Also, the expression of the T3-sensitive
gene hairless was not increased in the cerebral cortex of
A striking result from this study is that 90% of the pituitary D2 is found in Cga-expressing cells, namely, thyrotrophs and gonadotrophs (Figure 1). Given the absence of
any significant disruption in reproductive function in the
male or female Cga-cre D2KO mice, it is likely that the
bulk of this activity is in the thyrotrophs, although more
rigorous studies of gonadotroph function are required to
confirm this. The fact that nearly all pituitary D2 activity
is lost in the Cga-cre D2KO mouse is even more remarkable given the small fraction (⬃10%) of the mouse pituitary cells thought to be thyrotrophs. This result is in agreement with the high D2 activity in the transformed
thyrotroph cell line, T␣T1, and in situ studies of euthyroid
and hypothyroid rat pituitaries (28). We found no differences in D3 activity between Cga-cre D2KO and control
mice in pituitary, cerebral cortex, and hypothalamus.
These results indicate no compensatory changes occurred
in the expression of the T3-sensitive Dio3 gene as a consequence of D2 deficiency in the pituitary (Supplemental
Figure 2). There is a modestly lower (25%) D2 activity in
the cerebral cortex of the euthyroid Cga-cre D2KO mice,
but this difference is lost in the hypothyroid animals with
higher D2 expression. Interestingly, a similar decrease in
baseline cortical D2 was found by Fonseca et al (29), although the difference was not significant due to a high
variation in results. This may be due to low levels of Cgacre expression in brain as reported by Cushman et al (12).
In the adult Cga-cre D2KO mice, the serum T4 and T3
are normal, whereas the TSH is 1.5-fold higher in male
mice (Table 1). TSH was also increased to the same degree
in females, although this was not statistically significant
(P ⫽ .057). We interpret the absence of a serum T4 elevation in the adult Cga-cre D2KO mice in the present
study as a reflection of the relatively modest increase in the
serum TSH. Previous studies have shown that the normal
range for TSH can vary from 2- to 4-fold within a given
mouse strain (30). The absence of an increase in thyroid
weights in the KO mice supports this interpretation. The
normal serum T4 concentrations in the present study differ
from the elevated values found in the global D2KO mouse
and the recently reported mouse model where Cga-driven
cre was also used to inactivate thyrotroph and gonadotroph D2 (11, 29, 31, 32). The global D2KO mouse model
has a modest, but significant, TSH elevation (10, 17), but
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751
decrease in TSH biological activity),
even though T4 is elevated and thyroid
weights are increased by 60%. The
reason for these differences is not
clear. As mentioned, we found the serum T4 is elevated in the Cga-cre
D2KO mice during the neonatal period as is discussed below, with a transition to a normal T4 by P17 (Figure 5).
The inactivation of D2 in the Cgaexpressing thyrotrophs results in a
Figure 4. TSH␣ and TSH␤ mRNA expression in the pituitaries of euthyroid and hypothyroid Cgaresistance to the acute suppression of
cre D2KO and control mice. TSH␣ (A) and TSH␤ (B) are shown from euthyroid control (white
bars) and Cga-cre D2KO (black bars) male mice at time 0 and at 4 weeks after treatment with
TSH release by T4, but not by T3, in
1% sodium perchlorate and 0.1% MMI. Expression levels are normalized to cyclophilin A
the global D2KO hypothyroid mouse
expression and are expressed relative to euthyroid, control animals. Data are shown as mean ⫾
and in the recently reported euthyroid
SEM in arbitrary units (AU) and analyzed using Student’s unpaired t test. n ⫽ 3–5 animals/group.
thyrotroph-specific D2KO mouse
(Figure 2) (10, 13). The loss of thyrosurprisingly, the Fonseca et al (13) pituitary-specific
troph
D2
can
explain
this resistance to T4 and it is in agreeD2KO has net normal TSH concentrations (due to the
3-fold increase in TSH levels being mitigated by a 40% ment with earlier studies in the rat with an iopanoic acid
blockade of the D2 enzyme (5). A
novel and important result of this
study is that in addition to blocking the
down-regulation of TSH by T4, thyrotroph D2 deficiency also reduces the
TSH response to thyroid hormone deficiency by 3- to 4-fold. What is the
basis for this impaired response? One
might have predicted that because the
basal TSH is higher in the Cga-cre
D2KO mice, it would have remained
so during the response to reduced thyroid function. This would certainly be
the case if the increase was primarily
due to increased TRH, because mild
hypothyroidism is well known to sensitize the thyrotroph to the effects of
TRH (33). However, we found no decrease in D2 activity in the hypothalamus to explain an increase in TRH
synthesis or release (Figure 1C), no increase in basal TRH mRNA expression in the PVN (Supplemental Figure
5A), nor was there a decrease in TSH
bioactivity in euthyroid or hypothyroid mice that might suggest more subtle reductions in TRH in the Cga-cre
D2KO mice (Supplemental Figure 3).
Another plausible cause could be a deFigure 5. Serum thyroid hormone profiles of neonatal male and female control and Cga-cre
crease in TSH␤ expression in Cga-cre
D2KO mice. Serum TSH (A and B), T4 (C and D), and T3 (E and F) levels in male and female mice.
D2KO mice, but this was not found in
Control mice are indicated by open circles, whereas Cga-cre D2KO mice are represented by
either the euthyroid or hypothyroid
closed circles. We found significant difference by statistical analysis using two-way ANOVA.
*, P ⬍ .05; **, P ⬍ .01; ***, P ⬍ .001 followed by unpaired Student’s t test. n ⫽ 3–7 per group.
state (Figure 4).
752
Luongo et al
Pituitary Dio2 Role in Hypothyroidism
Endocrinology, February 2015, 156(2):745–754
36). Because a reduction in serum T4
is an early event during the development of hypothyroidism or iodine
deficiency (Figure 3), an increase in
TSH could then initiate an enhancement of rate-limiting steps in thyroid
hormone synthesis such as iodide
trapping and organification as well
as increase the T3 to T4 ratio in thyroid secretion (14).
A transiently reduced TSH response to hypothyroidism may result
from thyrotoxicosis during development. This has been observed in human newborns of mothers with
poorly controlled hyperthyroidism,
in rodents after excess T4 administration during early development,
and in the global D3-KO mouse, in
which both fetal and neonatal serum
T3 are elevated (26, 27, 37). A reduced sensitivity to a low-serum T4
in these examples derives from a delayed recovery of the sensitivity of
the hypothalamic-pituitary-thyroid
axis to low thyroid hormone levels.
In the Cga-cre D2KO mouse, howFigure 6. Deiodinase activity in neonatal male and female control and Cga-cre D2KO mice.
ever, we propose it may be a chronic
Pituitary D2 activity of male (A and B), cerebral cortical D2 activity (C and D), and hepatic D1
activity of male (E and F) male and female mice are shown. These are the same cohort of mice as
deficiency of intracellular thyroused in Figure 5, and statistical analyses are as for Figure 5.
troph T3 derived from T4, which reduces the sensitivity to a further loss
We speculate that one reason for the impaired increase of cellular T3 due to a decrease of circulating T4. Imporin serum TSH in response to the hypothyroid challenge in tantly, we did not find TRH deficiency or a decrease in
the Cga-cre D2KO mice may be the chronic absence of bioactivity of TSH (Supplemental Figure 3) in either euD2-mediated production of thyrotroph T3. Thus, the thyroid or hypothyroid mice, a secondary consequence of
basal saturation of nuclear thyroid hormone receptors TRH deficiency, unlike what was recently reported by
(TRs) is lower in the Cga-cre D2KO thyrotrophs than in Fonseca et al (Figure 2) (13). We also did not find higher
the D2-expressing controls, and this is reflected in the TSH subunit mRNA content in the Cga-cre D2KO mice
somewhat higher TSH. In the euthyroid rat pituitary, the despite the elevation in the circulating TSH. This suggests
TRs are over 80% occupied by T3 in the euthyroid state that it could be the T3-dependent regulation of TSH reand D2-catalyzed T3 in the pituitary accounts for approx- lease that is less responsive when intracellular pituitary T3
imately half of this TR-receptor bound T3 (4, 34). The is chronically reduced (38). We have previously shown
reduction in intranuclear T3 from this source increases the that this response is more rapid than inhibition of TSH
unoccupied TRs as much as 2-fold leading to an approx- mRNA synthesis in the hypothyroid rat (38).
imate doubling of TSH in the Cga-cre D2KO mice (Table
An intriguing result in newborn Cga-cre D2KO mice
1). As a result, the reduction in the contribution from D2- was the elevation of serum T4 during the early neonatal
generated pituitary T3 due to hypothyroidism should period, which did not occur in the adult mice (Figure 5). In
cause a 2-fold greater absolute increase in the unoccupied global D2KO mice, T4 has also been previously reported
nuclear TRs in the thyrotrophs of control mice than in to be elevated at P15 (39). The TSH increase in the Cga-cre
those with D2 deficiency. The unoccupied TRs bound to D2KO neonatal mice was only about 2-fold, not different
the TSH␤ gene in the thyrotrophs are thought to be the from that found in the adults. The biggest difference in
main driving force to both TSH synthesis and release (35, factors regulating the feedback loop between the neonates
doi: 10.1210/en.2014-1698
and adults is the much higher pituitary D2 levels in the
young mice (Figures 1 and 6). The cerebrocortical D2 was
identical at P5 and P17, suggesting the elevated D2 activity
in the neonatal mouse is specific to the pituitary. The larger
difference between the neonatal pituitary D2 in the controls and Cga-cre D2KO mice than in the adults may explain their higher serum T4 throughout the first 2 weeks of
life as a reflection of the absence of the T4-feedback
through the D2 pathway. Interestingly, the relative T4 elevation in Cga-cre D2KO neonates is greatest at P5, suggesting that it may also be present during the late fetal
period. The serum T4 and T3 increase in parallel in both
genotypes to several fold above that at P5 as the pituitary
D2 falls until P15 when they reach the normal neonatal
peak as previously noted. The increase in serum thyroid
hormones occurs independently of a change in TSH (Figure 5) in both genders and occurs at a time when hepatic
D1 is increasing (Figure 6, E and F). Thus, the increases in
thyroid hormones cannot be attributed to a decrease in the
deiodinative T4 or T3 clearance. The absence of a further
increase in TSH as T4 and T3 increase in both genotypes
suggests a gradual increase of the thyroid gland sensitivity
to TSH and a reduction in the importance of T4 per se to
the feedback regulation of TSH as pituitary D2 falls. These
results suggest that the T4 feedback at the thyrotroph level
is especially important in the early neonatal period in the
mouse and is attenuated by P17. In some sense, this is
reminiscent of the marked increase in TSH, and thyroid
hormone secretion, at birth in the neonatal human (40).
The persistent elevation of TSH, despite the rising serum
T4, establishes that neonatal suppression of the thyrotroph does not explain the impaired TSH response of the
adult Cga-cre D2KO mouse to hypothyroidism. Taken
together, the data argue that the higher serum T4 over the
neonatal period in the Cga-cre D2KO mice is a result of an
autonomous TSH-mediated thyroidal stimulation due to
the reduced effectiveness of T4-mediated suppression in
the absence of D2.
Finally, it is well known that there are both transcriptional and posttranslational increases in D2, which can
buffer the adverse effects of a deficiency of circulating T4
in the CNS and attenuate the TSH response of the thyrotroph. Nonetheless, previous studies in the rat have demonstrated that the concentration of specifically bound nuclear T3 in the pituitary of the severely hypothyroid rat is
substantially reduced, despite the high D2, because of the
low-serum T4 (34). Although TRH has a critical role in the
regulation of thyroid function, these studies reveal the importance of D2 in modulating the physiological response
of the thyrotroph (41). We conclude that the capacity to
monitor both the prohormone T4 and the active hormone T3 is essential for the optimal response of the
endo.endojournals.org
753
vertebrate to the stress of chronic iodine deficiency or
primary hypothyroidism.
Acknowledgments
We thank Dr Stephen Huang and Ms Michelle Maynard for
assistance with the D3 assays. JP26 –26 and JP02 cells were a generous gift from Dr G. Vassart and S. Refetoff. Blastocyst injection
was performed by the BWH Transgenic Core.
Address all correspondence and requests for reprints to: P.
Reed Larsen, MD, Thyroid Section, Division of Endocrinology,
Diabetes, and Hypertension, Brigham and Women’s Hospital
and Harvard Medical School, Boston, MA 02115. E-mail:
[email protected].
Present address for A.M.: Department of Clinical and Experimental Medicine, Section of Endocrinology, University of Pisa,
56126-I Pisa, Italy.
This work was supported by National Institutes of Health
Grants R01DK36256, R01DK44128, T32DK007529, and
K01DK091403.
Disclosure Summary: The authors have nothing to disclose.
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