Dominant Role of Thyrotropin-Releasing Hormone in the Hypothalamic-Pituitary-Thyroid Axis
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JBC Papers in Press. Published on December 8, 2005 as Manuscript M511530200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M511530200
Dominant Role of Thyrotropin-Releasing Hormone in the
Hypothalamic-Pituitary-Thyroid Axis
Amisra A. Nikrodhanond1*, Tania M. Ortiga-Carvalho1,2*, Nobuyuki Shibusawa3,
Koshi Hashimoto3, Xiao Hui Liao1, Samuel Refetoff 1, Masanobu Yamada3,
Masatomo Mori3, and Fredric E. Wondisford1
1
From the Department of Medicine and the Committee on Molecular
Metabolism and Nutrition, Pritzker School of Medicine, The University of
Chicago, Chicago, Illinois, 60637, 2Instituto de Biofisica Carlos Chagas
Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 3
Department of Medicine and Molecular Science, Gunma University
Graduate School of Medicine, Maebashi, Gunma, Japan, *joint first authors
Running Title: Role of TRH in the Thyroid Axis
Address correspondence to: Fredric E Wondisford, Division of Metabolism,
Departments of Pediatrics and Medicine, Johns Hopkins Medical Institutes, Baltimore,
MD; Email: fwondisford@jhmi.edu
Hypothalamic thyrotropin-releasing hormone Thyroid hormones (THs) thyroxine (T4)
(TRH) stimulates thyroid-stimulating hormone and its biologically active derivative
(TSH) secretion from the anterior pituitary. TSH triiodothyronine (T3) play a critical role in
then initiates thyroid hormone (TH) synthesis development, growth, and cellular metabolism. T3
and release from the thyroid gland. While acts by binding to specific nuclear receptor
opposing TRH and TH inputs regulate the proteins, which modify gene transcription. Free
hypothalamic-pituitary-thyroid (HPT) axis, TH TH levels are regulated by negative feedback at
negative feedback is thought to be the primary the hypothalamic thyrotropin-releasing hormone
regulator. This hypothesis, however, has yet to be (TRH) neuron and pituitary thyrotroph. The
proven in vivo. To elucidate the relative synthesis of TRH, produced in the hypothalamus,
importance of TRH and TH in regulating the and the α and β subunits of thyrotropin (TSH,
HPT axis, we have generated mice, which lack thyroid-stimulating hormone), produced in the
either TRH, the β isoforms of TH receptors (TRβ anterior lobe of the pituitary, are inhibited at the
KO), or both (double KO). TRβ KO mice have transcriptional level by TH (1, 2). TH also inhibits
significantly higher TH and TSH levels compared post-translational modification of TSH as well as
to wild-type mice, in contrast to double KO mice, TSH release (1). Furthermore, TH also modulates
which have reduced TH and TSH levels. TSH expression by altering pituitary levels of
Unexpectedly, hypothyroid double KO mice also TRH receptors, and thyroid hormone receptors
failed to mount a significant rise in serum TSH (TRs) (3, 4). While hypothalamic TRH stimulates
levels, and pituitary TSH immunostaining was TSH synthesis and release, TH negative feedback
markedly reduced compared to all other at the pituitary is believed to be the most important
hypothyroid mouse genotypes. This impaired physiological regulator of serum TSH levels (5).
TSH response, however, was not due to a reduced Thyroid hormones have both genomic and
number of pituitary thyrotrophs because non-genomic effects (6, 7), although most workers
thyrotroph cell number, as assessed by counting believe that thyroid hormones act predominantly
TSH immunopositive cells, was restored after through a genomic mechanism. Genomic action is
chronic TRH treatment. Thus, TRH is absolutely mediated by different TR isoforms, which are
required for both TSH and TH synthesis but is members of the nuclear receptor superfamily of
not necessary for thyrotroph cell development. ligand modulated transcriptional factors (8).
Alternative splicing and alternative transcription
1
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.initiation of two genes produce all known ligand- loci (TRH-/-TRβ-/-, double KO). Genotyping of
binding TR isoforms: TRα1, TRβ1, TRβ2 and TRH and TRβ gene KO animals was performed on
TRβ3. The expression and regulation of the TRs tail extracts of genomic DNA, using Southern blot
vary with isoform and tissue type. Whereas both analysis and polymerase chain reaction (PCR) as
TRα1 and TRβ1 are expressed in most cell types, described previously (26, 27).
TRβ2 mRNA is selectively expressed in the anterior Animals were maintained under light/dark
pituitary, specific areas of the hypothalamus, and in cycles of 12:12 hours (lights on at 0600 h),
the developing brain and middle ear (9-11). Mice weaned after 21 days, and fed chow and water ad
deficient in either TRα or TRβ display unique libitum. All mice used in these experiments were
phenotypes, suggesting that different TR isoforms of the same mixed background strain
have unique regulatory roles (12-20). TH effects on (129svj/C57BL/6), and wild-type (WT) littermate
negative feedback of the hypothalamic-pituitary- mice served as normal controls. All animal
thyroid (HPT) axis are mediated, mostly, by the β2 experiments were performed according to the
isoform of TR (15, 16, 18-20). National Institute of Health (NIH) Guide for the
TRH is the major stimulator of TSH care and use of Laboratory Animals, and the
synthesis and release from the anterior pituitary (21, protocols were approved by the Institutional
22). Previous data have shown that TRH Animal Care and Use Committee at the University
administration to dams stimulated fetal pituitary and of Chicago.
thyroid function and that in vitro addition of TRH
activated embryonic pituitary cells. These data Serum thyroid hormone and TSH measurements
suggested that TRH is involved in regulation of Serum thyroid hormone levels (total T3
pituitary development and differentiation (23, 24). and T4) were measured by solid phase
In mice deficient in TRH (TRH KO mice), however, radioimmunoassay (Coat-A-Count, DPC). Mouse
a different conclusion was reached. Histological serum TSH levels were measured by a sensitive
examination of the embryonic anterior pituitary of heterologous radioimmunoassay, as described
these KO mice revealed that the number of TSH-β previously (35). Serum TSH bioactivity was
immunopositive cells was not affected in pups born determined by measuring cyclic adenosine
to TRH deficient mothers (25). Thus, neither monophosphate, (cAMP) levels in a Chinese
embryonic nor maternal TRH is required for normal hamster ovary cell line stably transfected with a
development of pituitary thyrotrophs. Adult animals human TSH receptor cDNA, as previously
lacking TRH have slightly increased levels of TSH described (36, 37). Serum was depleted of TSH
and lower levels of thyroid hormones, suggesting a by treatment of mice with 5 µg L-T4 for 10 days
decrease in the bioactivity of TSH and central and used as a blank in all assays (35). Mouse
hypothyroidism (26). To understand the relative serum TSH standard was produced by rendering
importance of TRH and thyroid hormone feedback WT mice hypothyroid as described (35).
in regulation of the HPT axis, we studied TRH, TRβ Hypothyroid mouse serum was diluted in TSH
depleted serum to generate the standard curve.
and both TRH and TRβ KO mouse models.
The standard curve was linear over the entire
concentration range, indicating a lack of an
Experimental Procedures
interfering substance. This serum was also, not
contaminated with other pituitary glycoproteins
Generation of TRH and TR-β knockout (KO) mice.
present in pituitary extracts. The TSH standard
TRH KO and complete TRβ KO mice were had identical immunological and biological
generated as described previously (26, 27). The two activity as serum TSH derived from congenitally
lines were crossed to generate heterozygous mice for hypothyroid Pax-8 KO mice (37).
both the TRH and TRβ KO loci. These Thyroid hormone suppression was
heterozygous mice were then crossed to generate the induced in animals at 8 weeks of age with a low
following genotypes: 1) normal mice (TRH+/+TRβ+/+, iodine diet (LoI) containing 0.15% 5-propyl-2-
WT); 2) mice lacking the TRH locus (TRH-/-TRβ+/+, thiouracil, (PTU, Harlan Teklad Co., Madison,
TRH KO); 3) mice lacking the TRβ locus Wisconsin, USA) and 0.05% methimazole (MMI,
(TRH+/+TRβ-/-, TRβ KO), and 4) mice lacking both Sigma-Aldrich, St. Louis, Missouri, USA) in
2water. After 5 weeks, animals received daily TSH α-subunit, 5’-TCTCGCCGTCCTCCTCTC
subcutaneous injections of either vehicle or L-T3 CGTGCTT-3’ and 5’-AGTTGGTTCTGACAGC
(Sigma Corp) at a low (0.2 µg/100g body CTCGTG-3’ for the TSH β -subunit, 5’-
weight/day), medium (0.5 µ g/100g body TTCTCTCCTTCCTCCCATCCTTT-3’ and 5’-
weight/day), or high (1.0 µg/100g body weight/day) GGCTGGAGGGTCTGAGGG-3’ for TRα1 and
dose for 7 days each. The LoI/PTU diet and MMI in 5’-CGGCTACCACATCCAAGGAA-3’ and 5’-
water were given throughout the L-T3 treatment GCTGGAATTACCGCGGCT-3’ for the 18S
period. Animals were sacrificed 24 hours after the ribosomal subunit.
last injection of L-T3. A group of animals was also Relative mRNA levels (2Δ Ct) were
sacrificed after LoI/PTU diet, and these pituitaries determined by comparing the PCR cycle threshold
subjected immunohistochemistry as described (Ct) between groups. The purity of the PCR
below. products was checked by analyzing the melting
curves. Each sample was measured in duplicate
TRH Treatment and each experiment was repeated at least 3 times.
Animals were given a LoI/PTU diet and MMI in All the results were expressed relative to WT
water for a total of 24 days to induce expression considered as 100%.
hypothyroidism. After 14 days of LoI/PTU and
MMI treatment, placebo pellets or pellets containing In situ hybridization
10 mg of TRH (Innovative Research of America) In situ hybridization histochemistry was
were implanted subcutaneously. Blood samples performed following a previously described
were collected from the orbital vein 0, 5, and 10 protocol (38) with adjustments described below.
days after pellet implantation, and TSH assayed as Animals were anesthetized and perfused
described above. Animals were sacrificed and intracardially following protocol with 10%
pituitary glands subjected to immunohistochemistry phosphate-buffered formalin (Fisher Scientific).
as described below. Brains were removed to a 10% sucrose solution in
10% phosphate-buffered formalin overnight at 4ºC
RNA analysis with gentle shaking to be sectioned the next day.
Total RNA was extracted by standard Coronal sections 30 µm thick were cut using a
methodology (TRIzol Reagent, Life Technologies, Leica SM 2000R sliding microtome (Leica
Invitrogen). For quantitative real-time reverse Microsystems, Bannockburn, Illinois, USA).
transcriptase PCR (real-time RT-PCR) analysis, Sections were collected free-floating in cold
reverse transcription (RT) was carried out on 2 µg of phosphate buffered saline (PBS) treated with
total pituitary RNA. Real-time RT-PCR analyses DEPC and mounted as previously described onto
were performed in a fluorescent temperature cycler Fisher Scientific Superfrost Plus slides (38).
(MyiQ, single color Real-time PCR Detection After drying, slides were treated with a 0.001%
System, Bio-Rad Laboratories) according to the proteinase K solution. Hybridization was carried
recommendations of the manufacturer. Briefly, after out overnight (16-18 hours) at 65ºC on a slide
initial denaturation at 50oC for 2 min and 95oC for warmer using 1 x 107 cpm of probe per ml of
10 min, reactions were cycled 40 times using the hybridization solution prepared as per protocol.
following parameters for all genes studied: 95oC for To prepare the riboprobe used in
15 seconds, 60oC for 30 seconds, and 72oC for 30 hybridization, bases 272 to 629 of exon 3 of the
seconds. SYBR Green I (Bio-Rad) fluorescence was mouse thyrotropin releasing hormone gene (NCBI,
detected at the end of each cycle to monitor the accession# NM_009426) was PCR amplified from
amount of PCR product formed during that cycle. WT mouse genomic DNA using the forward
Primers used for the amplification of cDNAs of primer, 5’- TCCTG G A T C C C A A A A C G
interest were synthesized by IDT (Integrated DNA CCAGCAT-3’, with the change of a single base to
Technologies). The sequence of the forward and create an internal Bam HI site. The reverse
reverse primers was, respectively: 5’- primer, 5’- AGCTTCTTTGG A G C T
GTGTATGGGCTGTTGCTTCTCC-3’ and 5’- CAGGATCTA- 3’, contained an internal SacI site.
GCACTCCGTATGATTCTCCACTCTG-3’ for the The PCR product was ligated into pGEMT
3(Promega Corp.) and sequenced. To linearize the stained with nickel enhancement or DAB (Vector
vector for in vitro transcription, 5 µg of DNA was Labs) and hematoxylin to visualize TSH positive
digested with SalI and phenol/chloroform extracted. cells, or thyrotrophs, for the purpose of counting
Transcription was completed using 1 µg of DNA cell number. Three non-overlapping areas in the
template, with the Promega Riboprobe in vitro anterior pituitary of 3 to 5 animals per group were
Transcription System, which included 35S-UTP. observed under 400x magnification and the images
Post-hybridization washes and film signal captured (Image Pro Plus). The total number of
detection of sections followed protocol with cells and the number of TSH-β positively stained
exposure of slides for 3 days to Kodak BioMax-MR cells were counted for the entire field-of-view of
film (Eastman Kodak Co.). Slides were then coated each area. To determine the relative amount of
with NTB emulsion (Eastman Kodak) and protected TSH-β immunopositive cells, the ratio of the
from light in an aluminum foil-covered microscope number of TSH-positively-stained cells to the total
slide box at 4°C for 3 weeks. Images of sections number of cells for each field-of-view was
after development were captured and quantitated calculated.
using the software Image Pro Plus, version 4.5.1.22
for Windows (Media Cybernetics, Inc., Silver Histology
Spring), and an Olympus BH2-RFCA microscope Thyroid glands were excised, washed once
(Olympus America Inc.) equipped with a Sony with PBS, and then fixed in 10% phosphate-
DXC-960MD color analog video camera (Sony buffered formalin and embedded in paraffin.
Corp). After subtracting background measurements, Sections 6 µm thick were prepared and stained
the mean values for the PVN and for the lateral with hematoxylin/eosin (H&E).
hypothalamus of each animal were calculated. The
TRH expression within PVN is regulated by T3 (39- Statistical analysis
41). Ratios were then calculated of the mean PVN Data are reported as means ± SEM. One-
value to the mean lateral hypothalamic (area not way ANOVA followed by Student-Newman-
affected by T3) value for each animal to determine Keuls multiple comparisons test was employed for
the relative degree of difference in TRH mRNA assessment of significance when comparisons
expression of the TRH-regulated PVN relative to the were made within the same genotype. Two-way
lateral hypothalamus. ANOVA was employed when mice of different
genotypes and treatment were compared
Immunohistochemistry (GraphPad Prism, GraphPad Software, Inc.).
Animals were anesthetized and then All experiments were repeated at least 3 times,
perfused intracardially with 4% paraformaldehyde. except the experiment with TRH treatment that
Pituitaries were excised and post-fixed overnight at was repeated twice.
4ºC with 4% paraformaldehyde containing 10%
sucrose and gently shaken. Tissues were embedded RESULTS
in paraffin and sectioned in 3µ m thick slices
sagittally. Sections were prepared and blocked with To compare the relative importance of
2% normal goat serum (Vector Labs) for 1 hour at TRH and TRβ in feedback regulation of the HPT
room temperature and hybridized with a 1:1000 axis, we generated three groups of KO mice each
dilution of rabbit anti-rat TSH-β antibody obtained deficient in either or both protein(s). To establish
from Dr. A.F. Parlow of the National Hormone & that double KO mice were not expressing TRH,
Peptide Program (rTSH-β-IC-1, lot# AFP1274789) we measured hypothalamic TRH mRNA using in
overnight (16-18 hours) at 4ºC. After sections were situ hybridization histochemistry (Figure 1A). As
washed and biotinylated goat, anti-rabbit secondary expected, TRH mRNA was absent in TRH KO
antibody (Vector Labs) applied for 1 hour at room and double KO mice, while TRβ KO mice
temperature, sections were washed. Avidin-biotin- demonstrated an increase in the TRH expression in
horseradish peroxidase, provided in the Standard the paraventricular nucleus (PVN) when compared
Elite Vectastain ABC kit (Vector Labs), was applied to WT animals (Figure 1B). The latter result is
according to standard protocol. Sections were DAB consistent with a defect in negative T3 regulation
4of the TRH neuron as reported previously (20). correcting for serum TSH levels (cAMP/TSH
Double KO mice were born with no gross anatomic ratio). After this correction, TSH bioactivity was
or functional abnormalities and were viable through decreased in all KO groups when compared to the
adulthood. Both male and female mice displayed WT mice, being significantly decreased in TRH
normal fertility. and double KO mice. Another measure of TSH
As previously reported, the absence of TRβ bioactivity is the serum T4/TSH ratio (ref. 28 and
results in a defect in negative feedback regulation of Refetoff S., unpublished results). The T4/TSH
the HPT axis resulting in higher TH levels in these ratio (Figure 3A, lower panel) of all KO groups
mice. As shown in Figure 2, total serum T3 and T4 showed a significantly decreased ratio compared
levels were highest in the TRβ KO mice, reaching to WT animals. This assay, however,
statistical significance versus WT mice (15, 16, 27). underestimates TSH bioactivity when TSH values
Consistent with previous reports, TRH KO mice are markedly elevated due to the linear-log
presented with decreased serum T4 levels when relationship between changes in T4 and TSH. A
compared to WT animals (Pdemonstrated a resistance to L-T3 suppression at the been demonstrated in TR KO animals that TRβ2 is
highest dose (2, 27). the dominant isoform mediating T3-negative
Like serum TSH, TSH subunit mRNA levels regulation of TSH subunit gene expression in the
in double KO mice were not significantly elevated pituitary and that TRβ 2 is the main isoform
after PTU treatment (Figure 4C and 4D). TSH α regulating TRH gene expression in the
and β subunits mRNA responded to PTU treatment hypothalamus (16, 20). The importance of TRβ2
in all groups (except double KO animals). However, in regulating the HPT axis may reflect a higher
the magnitude of TSH-α subunit mRNA response expression level of this isoform in tissues
was lower when compared to the TSH-β subunit regulating the HPT axis, since it has been shown
mRNA levels (Figure 4C and 4D, respectively). that somatic gene transfer of either TRβ1 or TRβ2
To define the number of TSH-producing rescues the hypothalamic defect in TRH negative
cells in the pituitary, TSH-β immunopositive cells regulation in TRβ KO animals (29). In contrast,
were quantitated in all groups after induction of TRα1 deletion alone or in combination with α2
hypothyroidism (Figure 5). After 35 days of causes a mild central hypothyroidism perhaps due
LoI/PTU + MMI treatment, the number of TSH-β to a regulatory or developmental defect in the HPT
immunopositive cells, corrected for total cells in the axis (13, 14, 30).
field, was similar in WT and TRβ KO mice (Figure Hypothyroidism is necessary but not
5B, lower panel). In contrast, the number of TSH-β sufficient to upregulate the HPT axis. This was
immunopositive cells was somewhat lower in TRH suggested by a study of patients with central
KO mice and significantly lower in the double KO hypothyroidism caused by hypothalamic
mice. Fewer TSH-β immunopositive cells were dysfunction and definitively shown in mice
observed throughout the anterior lobe of the double lacking TRH (26, 31). Hypothalamic TRH
KO animals (Pthe TSH bioactivity. TSH bioactivity was measured which demonstrated that either TRH or the
directly by determining cAMP generation in an in absence of TH was sufficient to mediate an
vitro TSH bioassay. Although serum TSH levels increase in TSH subunit gene transcription (2, 34).
were higher in all KO groups, cAMP/TSH ratios (a To explore further the mechanism for the
measure of TSH bioactivity) were decreased when decreased serum TSH in double KO mice, we
compared to WT animals (Figure 3A). This finding measured the number of TSH-β immunopositive
most likely illustrates the critical role that TRH cells in pituitary sections. Compared to WT
plays in TSH glycosylation in the anterior pituitary, animals, the number of TSH-β immunopositive
as previously reported (31-33). We did not observe cells was similar in TRβ KO mice, somewhat
a goiter TRβ KO animals even though their absolute decreased (but not significantly) in TRH KO mice,
TSH level was elevated (Figure 3B and C). This and markedly decreased in double KO animals
may be due to the age of the mice used in our study during hypothyroidism. Since the number of
– we find that goiter in TRβ KO animals is age- detectable TSH-β immunopositive cells was
dependent. In contrast, the thyroid gland was decreased in the double KO during induced
smaller in TRH and double KO mice, suggesting hypothyroidism (Figure 5A, B), these data could
that the increased serum TSH levels in these mice suggest that the combination of TRH and TH (via
could not compensate for a reduction in serum TSH TRβ) are required for thyrotroph cell development
bioactivity. and/or maintenance.
We next studied negative regulation of the To confirm this hypothesis, we evaluated
central axis by T3 after animals were rendered the response of KO animals to TRH stimulation
hypothyroid. As previously reported (27), TRβ KO (Figure 6). Slow-release TRH or placebo pellets
animals were less responsive to T3 such that at the were implanted in hypothyroid animals. After this
highest T3 dose TSH was still markedly elevated treatment, no significant differences in either
(Figure 4A, inset). Double KO mice behaved as TSH-β immunopositive cells or serum TSH levels
TRβ KO animals even though TSH levels were were found among the groups, indicating that the
much lower at the beginning of T3-treatment (see defect in double KO mice was corrected after TRH
below). In contrast, the responsiveness of the t r e a t m e n t . These results indicate that the
thyrotroph to exogenous T3 was increased in TRH decreased serum TSH values observed in the
KO mice; TSH values returned rapidly with the double KO animals during hypothyroidism are due
smallest T3 dose. This result suggested that the to decreased TSH synthesis and not due to a
thyrotroph might be more sensitive to T3 reduction in thyrotroph cell number.
negative feedback without hypothalamic TRH Others have shown that TRH is not
input. The most unexpected result in this study was physiologically required for the proliferation or
that thyrotrophs from double KO mice failed to differentiation of embryonic thyrotrophs. After
respond to hypothyroidism. This experiment was birth, however, the number of TSH-β
designed to ensure that all the groups became immunopositive cells decreases showing the
equally hypothyroid before administration of T3 (T4 importance of TRH in maintenance of normal
was undetectable in all groups treated with LoI/PTU postnatal functions of the pituitary thyrotrophs
and MMI). Serum TSH levels were at least 50-fold (25). Our results show that TRH KO mice
lower, and TSH subunits mRNA levels at least 2- respond normally or nearly normally to
fold lower, in double KO mice during the hypothyroidism but that double KO mice display a
hypothyroid phase of the experiment. Given that we significantly impaired response. Serum TSH both
observed appropriately increased serum TSH levels failed to increase normally after hypothyroidism as
in WT, TRH KO and TRβ KO mice, we concluded well as suppress normally after T3 administration.
that the presence of both TRβ and TRH is necessary These data suggest a previously unrecognized
for a normal thyrotroph response during interaction between TRH and TH signaling
hypothyroidism suggesting that unliganded TRβ pathways in mediating the hypothyroid TSH
stimulates TSH subunit gene expression (Figure 4). response. We can also speculate that TRH
Support for these findings can be found in signaling may enhance stimulation by the
previous studies of primary thyrotroph cell cultures,
7unliganded TRβ by an unknown cross-talk lacking TRH, even when negative feedback is also
mechanism. disrupted (double KO mice). This defect in
double KO mice reflects a marked decrease in
In conclusion, TRH is critical for normal TSH synthesis, which was reversed by chronic
regulation of the HPT axis. TRH absence causes TRH stimulation. Although hypothyroidism is
central hypothyroidism in mice due to the synthesis known to markedly increase TSH synthesis at a
of biologically less active TSH. When challenged transcriptional level, these results indicate an
with primary hypothyroidism, however, the central unexpected, dominant role for TRH in regulating
axis is also unable to respond normally in mice the HPT axis in the basal and hypothyroid state.
REFERENCES
1. Cohen, R.C., and Wondisford F.E. (2005) In Werner and Ingbar’s The Thyroid: a fundamental
and clinical text, 9th edition. Braverman, L.E. and Utiger, R.D. Williams & Wilkins, Philadelphia, USA.
159-175.
2. Shupnik, M.A, Chin, W.W., Habener, J.F., Ridgway, E.C. 1986 Transcriptional regulation of the
thyrotropin subunit genes by thyroid hormone. J Biol Chem. 260:2900-2903.
3. Lean, A.D., Ferland, L., Drouin, J., Kelly, P.A., Labrie, F. 1977. Modulation of pituitary
thyrotropin releasing hormone receptor levels by estrogens and thyroid hormones. Endocrinology.
100:1496-1504.
4. Hinkle, P. M., Goh, K.B.C. 1982. Regulation of thyrotropin releasing hormone receptors and
responses by L-triiodothyronine in dispersed rat pituitary cell cultures. Endocrinology. 110:1725-1731.
5. Shupnik MA. 2000. Thyroid hormone suppression of pituitary hormone gene expression. Rev
Endocr Metab Disord. 1:35-42
6. Bassett, J.H., Harvey, C.B., Williams, G.R. 2003 Mechanisms of thyroid hormone receptor-
specific nuclear and extra nuclear actions. Mol Cell Endocrinol. 213:1-11.
7. Scalan, T. S., Suchland, K. L., Hart, M. E., Chiellini, G., Huang, Y., Kruzich, P. J., Frascarelli, S.,
Crossley II, D.A., Bunzow, J. R., Ronca-Testoni, S., Lin, E. T., Hatton, D., Zucchi, R., Grandy, D. K.
2004. 3-iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nature
Medicine. 6:638-642.
8. Lazar, M.A. 2003. Thyroid hormone action: a binding contract. J. Clin. Invest. 112:497-499.
9. Bradley, D.J., Towle, H.C., and Young, W.S. 3rd. 1994. Alpha and beta thyroid hormone receptor
(TR) gene expression during auditory neurogenesis : evidence for TR isoform-specific transcriptional
regulation in vivo. Proc. Natl. Acad. Sci. U.S.A. 2:439-443.
10. Lechan, R.M., Yanping, Q.I., Jackson, I.M.D., and Madhavi, V. 1994. Identification of thyroid
hormone receptor isoforms in thyrotropin-releasing hormone neurons of the hypothalamic paraventricular
nucleus. Endocrinology. 135:92-100.
11. Hodin, R.A., Lazar, M.A., Wintman, B.I., Darling, D.S., Koenig, R.J., Larsen, P.R., Moore, D.D.,
Chin, W.W. 1989. Identification of a thyroid hormone receptor that is pituitary-specific. Science. 244:76-
79.
12. Flamant, F. and Samarut, J. 2003. Thyroid hormone receptors: lessons from knockout and knock-
in mutant mice. Trends in Endocr. Met. 14:85-90
13. Fraichard, A., Chassande, O., Plateroti, M., Roux, J.P., Trouillas, J., Dehay, C., Legrand, C.,
Gauthier, K., Kedinger, M., Malaval, L., Rousset, B., Samarut, J. 1997. The T3Ra gene encoding a
8thyroid hormone receptor is essential for post-natal development and thyroid hormone production. EMBO
J. 16:4412-4420.
14. Wikström, L., Johansson, C., Saltó, C., Barlow, C., Campos Barros, A. Baas, F., Forrest, D.,
Thorén, P., Vennström, B. 1998. Abnormal heart rate and body temperature in mice lacking thyroid
hormone receptor alpha1. EMBO J. 17:455-461.
15. Forrest, D., Erway, L.C., Ng, L., Altschuler, R., and Curran, T., 1996. Thyroid hormone receptor
beta is essential for development of auditory function. Nat. Genet. 13:354-357.
16. Abel, E.D., Boers, M.E., Pazos-Moura, C., Moura, E., Kaulbach, H., Zakaria, M., Lowell, B.,
Radovick, S., Liberman, M.C., Wondisford, F.E., 1999. Divergent roles for thyroid hormone receptor
beta isoforms in the endocrine axis and auditory system. J. Clin. Invest. 104:291-300.
17. Ng, L., Rüsch, A., Amma, L.L., Nordström, Erway, L.C., Vennström, B., Forrest, D. 2001.
Suppression of the deafness and thyroid dysfunction in Thrb-null mice by an independent mutation in the
Thra thyroid hormone receptor alpha gene. Hum. Mol. Gen. 10:2701-2708.
18. Gauthier, K., Chassande, O., Plateroti, M., Roux, J.-P., Legrand, C., Pain, B., Rousset, B., Weiss,
R., Trouillas, J., Samarut, J. 1999. Different functions for the thyroid hormone receptors TRalpha and
TRbeta in the control of thyroid hormone production and post-natal development. EMBO J. 18:623-631.
19. Weiss, R.E., Forrest, D., Pohlenz, J., Cua, K., Curran, T., Refetoff, S. 1997. Thyrotropin
regulation by thyroid hormone in thyroid hormone receptor beta-deficient mice. Endocrinology.
138:3624-3629.
20. Abel, E.D., Ahima, R.S., Boers, M.E., Elmquist, J.K. and Wondisford, F.E. 2001. Critical role
for thyroid hormone receptor beta2 in the regulation of paraventricular thyrotropin-releasing hormone
neurons. J. Clin Invest. 107:1017-1023.
21. Harris, A.R., Christianson, D., Smith, M.S., Fang, S.L., Braverman, L.E., Vagenakis, A.G. 1978.
The physiological role of thyrotopin-releasing hormone in the regulation of thyroid-stimulating hormone
and prolactin secretion in the rat. J. Clin. Invest. 61:441-448.
22. Steinfelder, H.J., Hauser, P., Nakayama, Y., Radovick, S., McClaskey, J.H., Taylor, T.,
Weintraub, B.D., Wondisford, F.E. 1991. Thyrotropin-releasing hormone regulation of human TSHB
expression: role of a pituitary-specific transcription factor (Pit-1/GHF-1) and potential interaction with a
thyroid hormone-inhibitory element. Proc. Natl. Acad. Sci. USA 88:3130-3134.
23. D’Angelo, S.A. and Wall, N.R. 1971. Maternal-fetal endocrine interrelations: effects of synthetic
thyrotrophin releasing hormone (TRH) on the fetal pituitary-thyroid system of the rat.
Neuroendocrinology 9:197-206.
24. Kojima, A. and Hershman, J.M. 1974. Effects of thyrotropin-releasing hormone (TRH) in
maternal, fetal and newborn rats. Endocrinology. 94:1133-1138.
25. Shibusawa, N., Yamada, M., Hirato, J., Monden, T., Satoh, T., Mori, M. 2000. Requirement of
thyrotropin-releasing hormone for the postnatal functions of pituitary thyrotrophs: ontogeny study of
congenital tertiary hypothyroidism in mice. Mol. Endocrinol. 14:137-146.
26. Yamada, M., Saga, Y., Shibusawa, N., Hirato, J., Murakami, M., Iwasaki, T., Hashimoto, K.,
Satoh, T., Wakabayashi, K., Taketo, M.M., Mori, M. 1997. Tertiary hypothyroidism and hyperglycemia
in mice with targeted disruption of the thyrotropin-releasing hormone gene. Proc. Natl. Acad. Sci. USA
94:10862-10867.
27. Shibusawa, N., Hashimoto, K., Nikrodhanond, A.A., Liberman, M.C., Applebury, M.L., Liao,
X.H., Robbins, J.T., Refetoff, S., Cohen, R.N. and Wondisford, F.E. 2003. Thyroid hormone action in
the absence of thyroid hormone receptor DNA-binding in vivo. J. Clin. Invest. 112: 588-597.
28. Lado-Abeal, J., Dumitrescu, A.M., Liao, X.H., Cohen, R.N., Pohlenz, J., Weiss, R.E., Lebrethon,
M.C., Verloses, A., Refetoff, S. 2005. A de novo mutation in an already mutant nucleotide of the thyroid
hormone receptor β gene perpetuated resistance to thyroid hormone. J. of Clin. Endocrinol and Metab.
90:1760-1767.
929. Dupre, S. M., Guissouma, H., Flamant, F., Seugnet, I., Scanlan, T. S., Baxter, J.D., Samarut, J.,
Demeneix, B. A., Becker, N. 2004 Both thyroid hormone receptor (TR)β1 and TRβ2 isoforms contribute
to the regulation of hypothalamic thyrotropin-releasing hormone. Endocrinol. 145:2337-2345.
30. Macchia, P.E., Takeuchi, Y., Kawai, T., Cua, K., Gauthier, K., Chassandre, O., Seo, H., Hayashi,
Y., Samarut, J., Murata, Y., Weiss, R.E., Referoff, S. 2001. Increased sensitivity to thyroid hormone in
mice with complete deficiency of thyroid hormone receptor alpha. Proc Natl Acad Sci. 98:349-254.
31. Taylor, T., and Weintraub, B.D. 1989. Altered thyrotropin (TSH) carbohydrate structures in
hypothalamic hypothyroidism created by paraventricular nuclear lesions are corrected by in vivo TSH-
releasing hormone administration. Endocrinology. 125:2198-2203.
32. Beck-Peccoz, P., Amr, S., Menezes,-Ferreira, M.M., Faglia, G., Weintraub, B.D. 1985. Decreased
receptor binding of biologically inactive thyrotropin in central hypothyroidism. Effect of treatment with
thyrotropin-releasing hormone. N. Engl. J. Med. 312:1085-1090.
33. Persani, L., Ferretti, E., Borgato, S., Faglia, G., Beck-Peccoz, P. 2000. Circulating thyrotropin
bioactivity in sporadic central hypothyroidism. J. Clin. Endocrinol. Metab. 85:3631-3635.
34. Shupnik, M.A., Greenspan S.L., Ridgway E.C. 1986 Transcriptional regulation of thyrotropin
subunit genes by thyrotropin-releasing hormone and dopamine in pituitary cell culture. J Biol Chem.
261:12675-12679
35. Pohlenz, J., Maqueem, A., Cua, K., Weiss, R.E., Van Sande, J., Refetoff, S. 1999. Improved
radioimmunoassay for measurement of mouse thyrotropin in serum: strain differences in thyrotropin
concentration and thyrotroph sensitivity to thyroid hormone. Thyroid. 9:1265-1271.
36. Perret, J., Lugate, M., Libert, F., Gerard, C., Dumont, J.E., Vassart, G., Parmentier, M. 1990.
Stable expression of the human TSH receptor in CHO cells and characterization of differentially
expressing clones. Bioch. Biophys. Res. Com. 171:1044-1050.
37. Moeller, L.C., Kimura, S., Kusakabe, T., Liao, X.-H., Van Sande, J., Refetof, S. 2003.
Hypothyroidism in thyroid transcription factor 1 haploinsufficiency is caused by reduced expression of
the thyroid-stimulating hormone receptor. Mol. Endocrinol. 17:2295-2302.
38. Simmons, D.M., Arriza, J.L., and Swanson, L.W. 1989. A complete protocol for in situ
hybridization of messenger RNAs in brain and other tissues with radio-labeled single-stranded RNA
probes. J. Histotech. 12:169-181.
39. Abel, E.D., Moura, E.G., Ahima, R.S., Campos-Barros, A., Pazos-Moura, C.C., Boers, M.E.,
Kaulbach, H.C., Forrest, D., Wondisford, F.E. 2003. Dominant inhibition of thyroid hormone action
selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of
thyrotropin by thyroid hormone. Mol. Endocrinol. 17:1767-1776.
40. Segerson, T.P., Kawe, J., Wolfe, H.C., Mobtaker, H., Wu, P. Jackson, I.M., Lechan, R.M. 1987
Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus.
Science. 238:78-80.
41. Dyess, E.M., Segerson, T.P., Liposits, Z., Paul, W.K., Kaplan, M.M., Wu, P., Jackson, I.M.,
Lechan, R.M. 1988 Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing
hormone gene expression in the hypothalamic paraventricular nucleus. Endocrinol. 123: 2291-2297.
Acknowledgments
This work was supported by grants from the National Institute of Health to F.E.W. (DK49126 and
DK53036) and the Diabetes Research and Training Center at the University of Chicago (DK20595). We
thank Sally Hall for technical assistance with the in situ hybridization histochemistry.
Abbreviations
HPT - hypothalamic-pituitary-thyroid; KO – knock out; LoI/PTU - low iodine diet containing 0.15% 5-
propyl-2-thiouracil; MMI – methimazole; PVN – paraventricular nucleus; T3 – triiodothyronine; T4 –
10thyroxine; TH -thyroid hormone; TR - thyroid hormone receptor; TRH - thyrotropin-releasing hormone;
TSH - thyroid-stimulating hormone
11FIGURE LEGENDS Figure 1. TRH mRNA level of WT, TRβ KO, TRH KO, and double KO mice. A. Representative dark- field photomicrographs showing pre-proTRH mRNA in the PVN of KO mice. B. Relative pre-proTRH mRNA expression. Seven to eight animals were evaluated in each group. No significant signal was detected in TRH KO or double KO mice. * ratio mathematically undefined Figure 2. Analysis of the hypothalamic-pituitary-thyroid axis in WT, TRβ KO, TRH KO, and double KO mice. Total serum T4, T3 and TSH levels. Data are shown as means ± SEM. *P
Figure 1
WT TRβ KO
A
PVN
Lat. Hypothalamus
TRH KO double KO
B
0.2 PVN
Relative Intensity
Lateral Hypothalamus
TRH mRNA
0.1
0.0
WT TRβ KO
TRH KO double KO
2 P = 0.002
Hypothalamus)
(PVN/Lateral
TRH mRNA
1
0 * *
WT TRβ KO
TRH KO double KO
13*
Figure 2 8
7
Serum T4 (µg/dl)
6
5
4
3 * *
2
1
0
WT TRH KO
TRβ KO double KO
100
*
Serum T3 (ng/dl)
75
50
25
0
WT TRH KO
TRβ KO double KO
450 *
400
Serum TSH (mU/L)
350
300
250
200
150 * *
100
50
0
WT TRH KO
TRβ KO double KO
14Figure 3
A 7.5
* B C
cAMP production
, pmol/tube
5.0
*
*
2.5
2.5
0.0 2.0
WT TRH KO WT
Thyroid area
TRβ KO double KO
(mm2)
1.5
1.0
*
*
0.03 0.5
TSH Bioactivity
* 0.0
0.02 * WT TRH KO
TRβ KO double KO
TRβ KO
0.01
0.00
WT TRH KO 30
TRβ KO double KO
Body Weight (g)
20
0.4
Serum T4/Serum TSH
TRHKO
10
0.3
0.2 0
WT TRH KO
TRβ KO double KO
0.1
* * *
0.0 double KO
WT TRH KO
TRβ KO double KO
15Figure 4
WT TRH KO
A double KO TRβ KO B
20000 1000 100000 Baseline
Serum TSH (mU/L)
PFigure 5
A After LoI/PTU diet
WT
300
B
IR TSH-β cells
200
40x 400x 100
40x
0
TRH KO WT TRH KO
TRβ KO double KO
750
Total nuclei
500
250
0
TRβ KO WT TRH KO
IR TSH-β cell/Total nuclei
TRβ KO double KO
0.7
0.6
0.5
0.4
0.3
0.2 *
0.1
Double KO 0.0
WT TRH KO
TRβ KO double KO
C
20000
TSH (mU/L)
15000
negative control
10000
5000
0
*
WT TRH KO
TRβ KO double KO
17Figure 6
A B Placebo TRH
600
450 PYou can also read
