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Originally published In Press as doi:10.1074/jbc.M511530200 on December 8, 2005

J. Biol. Chem., Vol. 281, Issue 8, 5000-5007, February 24, 2006
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Dominant Role of Thyrotropin-releasing Hormone in the Hypothalamic-Pituitary-Thyroid Axis*

Amisra A. Nikrodhanond{ddagger}1, Tania M. Ortiga-Carvalho{ddagger}§1, Nobuyuki Shibusawa, Koshi Hashimoto, Xiao Hui Liao{ddagger}, Samuel Refetoff{ddagger}, Masanobu Yamada, Masatomo Mori, and Fredric E. Wondisford{ddagger}2

From the {ddagger}Department of Medicine and the Committee on Molecular Metabolism and Nutrition, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637, §Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21000-000 Rio de Janeiro, Brazil, and the Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma 3-39-22, Japan

Received for publication, October 24, 2005 , and in revised form, December 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors (TRbeta KO), or both (double KO). TRbeta knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Thyroid hormone (TH)3 thyroxine (T4) and its biologically active derivative triiodothyronine (T3) play a critical role in development, growth, and cellular metabolism. T3 acts by binding to specific nuclear receptor proteins that modify gene transcription. Free TH levels are regulated by negative feedback at the hypothalamic thyrotropin-releasing hormone (TRH) neuron and pituitary thyrotroph. The synthesis of TRH, produced in the hypothalamus, and the {alpha} and beta subunits of thyrotropin (TSH, thyroid-stimulating hormone), produced in the anterior lobe of the pituitary, are inhibited at the transcriptional level by TH (1, 2). TH also inhibits post-translational modification of TSH as well as TSH release (1). Furthermore, TH also modulates TSH expression by altering pituitary levels of TRH receptors and thyroid hormone receptors (TRs) (3, 4). Although hypothalamic TRH stimulates TSH synthesis and release, TH negative feedback at the pituitary is believed to be the most important physiological regulator of serum TSH levels (5).

Thyroid hormones have both genomic and non-genomic effects (6, 7), although most workers believe that thyroid hormones act predominantly through a genomic mechanism. Genomic action is mediated by different TR isoforms that are members of the nuclear receptor superfamily of ligand-modulated transcriptional factors (8). Alternative splicing and alternative transcription initiation of two genes produce all known ligand binding TR isoforms: TR{alpha}1, TRbeta1, TRbeta2, and TRbeta3. The expression and regulation of the TRs vary with isoform and tissue type. Whereas both TR{alpha}1 and TRbeta1 are expressed in most cell types, TRbeta2 mRNA is selectively expressed in the anterior pituitary, specific areas of the hypothalamus, and in the developing brain and middle ear (9–11). Mice deficient in either TR{alpha} or TRbeta display unique phenotypes, suggesting that different TR isoforms have unique regulatory roles (12–20). TH effects on negative feedback of the hypothalamic-pituitary-thyroid (HPT) axis are mediated, mostly, by the beta2 isoform of TR (15, 16, 18–20).

TRH is the major stimulator of TSH synthesis and release from the anterior pituitary (21, 22). Previous data have shown that TRH administration to dams stimulated fetal pituitary and thyroid function and that in vitro addition of TRH activated embryonic pituitary cells. These data suggested that TRH is involved in regulation of pituitary development and differentiation (23, 24). In mice deficient in TRH (TRH KO mice), however, a different conclusion was reached. Histological examination of the embryonic anterior pituitary of these KO mice revealed that the number of TSH-beta immunopositive cells was not affected in pups born to TRH-deficient mothers (25). Thus, neither embryonic nor maternal TRH is required for normal development of pituitary thyrotrophs. Adult animals lacking TRH have slightly increased levels of TSH and lower levels of thyroid hormones, suggesting a decrease in the bioactivity of TSH and central hypothyroidism (26). To understand the relative importance of TRH and thyroid hormone feedback in regulation of the HPT axis, we studied TRH, TR{alpha}, and both TRH and TRbeta KO mouse models.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of TRH and TRbeta Knock-out (KO) Mice—TRH KO and complete TRbeta KO mice were generated as described previously (26, 27). The two lines were crossed to generate heterozygous mice for both the TRH and TRbeta KO loci. These heterozygous mice were then crossed to generate the following genotypes: 1) normal mice (TRH+/+TRbeta+/+, WT), 2) mice lacking the TRH locus (TRH-/-TRbeta+/+, TRH KO), 3) mice lacking the TRbeta locus (TRH+/+TRbeta-/-, TRbeta KO), and 4) mice lacking both loci (TRH-/-TRbeta-/-, double KO). Genotyping of TRH and TRbeta gene KO animals was performed on tail extracts of genomic DNA, using Southern blot analysis and polymerase chain reaction (PCR) as described previously (26, 27).

Animals were maintained under light/dark cycles of 12:12 h (lights on at 0600 h), weaned after 21 days, and fed chow and water ad libitum. All mice used in these experiments were of the same mixed background strain (129svj/C57BL/6), and wild-type (WT) littermate mice served as normal controls. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Institutional Animal Care and Use Committee at the University of Chicago.

Serum Thyroid Hormone and TSH Measurements—Serum thyroid hormone levels (total T3 and T4) were measured by solid phase radioimmunoassay (Coat-A-Count; DPC). Mouse serum TSH levels were measured by a sensitive heterologous radioimmunoassay as described previously (35). Serum TSH bioactivity was determined by measuring cyclic adenosine monophosphate (cAMP) levels in a Chinese hamster ovary cell line stably transfected with a human TSH receptor cDNA as previously described (36, 37). Serum was depleted of TSH by treatment of mice with 5 µg of T4 for 10 days and used as a blank in all assays (35). Mouse serum TSH standard was produced by rendering WT mice hypothyroid as described (35). Hypothyroid mouse serum was diluted in TSH-depleted serum to generate the standard curve. The standard curve was linear over the entire concentration range, indicating lack of an interfering substance. This serum was also not contaminated with other pituitary glycoproteins present in pituitary extracts. The TSH standard had identical immunological and biological activity as serum TSH derived from congenitally hypothyroid Pax-8 KO mice (37).

Thyroid hormone suppression was induced in animals at 8 weeks of age with a low iodine diet (LoI) containing 0.15% 5-propyl-2-thiouracil, (PTU; Harlan Teklad Co., Madison, WI) and 0.05% methimazole (MMI; Sigma) in water. After 5 weeks, animals received daily subcutaneous injections of either vehicle or L-T3 (Sigma) at a low (0.2 µg/100 g of body weight/day), medium (0.5 µg/100 g body weight/day), or high (1.0 µg/100 g body weight/day) dose for 7 days each. The LoI/PTU diet and MMI in water were given throughout the L-T3 treatment period. Animals were sacrificed 24 h after the last injection of L-T3. A group of animals was also sacrificed after LoI/PTU diet, and these pituitaries were subjected to immunohistochemistry as described below.

TRH Treatment—Animals were given a LoI/PTU diet and MMI in water for a total of 24 days to induce hypothyroidism. After 14 days of LoI/PTU and MMI treatment, placebo pellets or pellets containing 10 mg of TRH (Innovative Research of America) were implanted subcutaneously. Blood samples were collected from the orbital vein 0, 5, and 10 days after pellet implantation, and TSH was assayed as described above. Animals were sacrificed and pituitary glands subjected to immunohistochemistry as described below.

RNA Analysis—Total RNA was extracted by standard methodology (TRIzol reagent; Invitrogen). For quantitative real-time reverse transcriptase PCR (real-time RT-PCR) analysis, reverse transcription (RT) was carried out on 2 µg of total pituitary RNA. Real-time RT-PCR analyses were performed in a fluorescent temperature cycler (MyiQ, single color real-time PCR detection system; Bio-Rad Laboratories) according to the recommendations of the manufacturer. Briefly, after initial denaturation at 50 °C for 2 min and 95 °C for 10 min, reactions were cycled 40 times using the following parameters for all genes studied: 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. SYBR Green I (Bio-Rad) fluorescence was detected at the end of each cycle to monitor the amount of PCR product formed during that cycle. Primers used for the amplification of cDNAs of interest were synthesized by IDT (Integrated DNA Technologies). The sequence of the forward and reverse primers was, respectively: 5'-GTGTATGGGCTGTTGCTTCTCC-3' and 5'-GCACTCCGTATGATTCTCCACTCTG-3' for the TSH {alpha}-subunit, 5'-TCTCGCCGTCCTCCTCTCCGTGCTT-3' and 5'-AGTTGGTTCTGACAGCCTCGTG-3' for the TSH beta-subunit, 5'-TTCTCTCCTTCCTCCCATCCTTT-3' and 5'-GGCTGGAGGGTCTGAGGG-3' for TR{alpha}1, and 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3' for the 18 S ribosomal subunit.

Relative mRNA levels (2{Delta}Ct) were determined by comparing the PCR cycle threshold (Ct) between groups. The purity of the PCR products was checked by analyzing the melting curves. Each sample was measured in duplicate, and each experiment was repeated at least three times. All the results were expressed relative to WT expression considered as 100%.

In Situ Hybridization—In situ hybridization histochemistry was performed following a previously described protocol (38) with adjustments described below. Animals were anesthetized and perfused intracardially following protocol with 10% phosphate-buffered formalin (Fisher Scientific). Brains were removed to a 10% sucrose solution in 10% phosphate-buffered formalin overnight at 4 °C with gentle shaking to be sectioned the next day. Coronal sections 30-µm thick were cut using a Leica SM 2000R sliding microtome (Leica Microsystems, Bannockburn, IL). Sections were collected free floating in cold phosphate-buffered saline treated with diethylpyrocarbonate and mounted as previously described onto Fisher Scientific Superfrost Plus slides (38). After drying, slides were treated with a 0.001% proteinase K solution. Hybridization was carried out overnight (16–18 h) at 65 °C on a slide warmer using 1 x 107 cpm of probe/ml of hybridization solution prepared according to the protocol.

To prepare the riboprobe used in hybridization, bases 272–629 of exon 3 of the mouse thyrotropin-releasing hormone gene (NCBI accession number NM_009426 [GenBank] ) were PCR amplified from WT mouse genomic DNA using the forward primer, 5'-TCCTGGATCCCAAAACGCCAGCAT-3', with the change of a single base to create an internal BamHI site. The reverse primer, 5'-AGCTTCTTTGGAGCTCAGGATCTA-3', contained an internal SacI site. The PCR product was ligated into pGEMT (Promega Corp.) and sequenced. To linearize the vector for in vitro transcription, 5 µg of DNA was digested with SalI and phenol/chloroform extracted. Transcription was completed using 1 µg of DNA template with the Promega riboprobe in vitro transcription system, which included 35S-UTP.

Post-hybridization washes and film signal detection of sections followed protocol with exposure of slides for 3 days to Kodak BioMax-MR film (Eastman Kodak Co.). Slides were then coated with NTB emulsion (Kodak) and protected from light in an aluminum foil-covered microscope slide box at 4 °C for 3 weeks. Images of sections after development were captured and quantitated using the software Image Pro Plus, version 4.5.1.22 [EC] for Windows (Media Cybernetics, Inc., Silver Spring, MD) and an Olympus BH2-RFCA microscope (Olympus America Inc.) equipped with a Sony DXC-960MD color analog video camera (Sony Corp). After subtracting background measurements, the mean values for the paraventricular nucleus (PVN) and for the lateral hypothalamus of each animal were calculated. The TRH expression within PVN is regulated by T3 (39–41). Ratios were then calculated of the mean PVN value to the mean lateral hypothalamic (area not affected by T3) value for each animal to determine the relative degree of difference in TRH mRNA expression of the TRH-regulated PVN relative to the lateral hypothalamus.


Figure 1
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  		FIGURE 1.
TRH mRNA level of WT, TRbeta 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.

 
Immunohistochemistry—Animals were anesthetized and then perfused intracardially with 4% paraformaldehyde. Pituitaries were excised and post-fixed overnight at 4 °C with 4% paraformaldehyde containing 10% sucrose and gently shaken. Tissues were embedded in paraffin and sectioned in 3-µm-thick slices sagittally. Sections were prepared and blocked with 2% normal goat serum (Vector Labs) for 1 h at room temperature and hybridized with a 1:1000 dilution of rabbit anti-rat TSH-beta antibody obtained from Dr. A. F. Parlow of the National Hormone & Peptide Program (rTSH-beta-IC-1, lot AFP1274789) overnight (16–18 h) at 4 °C. After sections were washed and biotinylated goat, anti-rabbit secondary antibody (Vector Labs) applied for 1 h at room temperature, sections were washed. Avidin-biotin-horseradish peroxidase, provided in the Standard Elite Vectastain ABC kit (Vector Labs), was applied according to standard protocol. Sections were diaminobenzidine tetrachloride stained with nickel enhancement or diaminobenzidine tetrachloride (Vector Labs) and hematoxylin to visualize TSH positive cells, or thyrotrophs, for the purpose of counting cell number. Three non-overlapping areas in the anterior pituitary of 3–5 animals/group were observed under x400 magnification and the images captured (Image Pro Plus). The total number of cells and the number of TSH-beta-positive-stained cells were counted for the entire field-of-view of each area. To determine the relative amount of TSH-beta immunopositive cells, the ratio of the number of TSH-positive-stained cells to the total number of cells for each field-of-view was calculated.


Figure 2
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  		FIGURE 2.
Analysis of the hypothalamic-pituitary-thyroid axis in WT, TRbeta KO, TRH KO, and double KO mice. Total serum T4,T3, and TSH levels. Data are shown as means ± S.E. *, p <0.01 or less. Five to fifteen animals were evaluated in each group.

 
Histology—Thyroid glands were excised, washed once with phosphate-buffered saline, and then fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections 6-µm thick were prepared and stained with hematoxylin/eosin.

Statistical Analysis—Data are reported as means ± S.E. One-way analysis of variance followed by Student-Newman-Keuls multiple comparisons test was employed for assessment of significance when comparisons were made within the same genotype. Two-way analysis of variance was employed when mice of different genotypes and treatment were compared (GraphPad Prism; GraphPad Software, Inc.). All experiments were repeated at least three times except for the experiment with TRH treatment, which was repeated twice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To compare the relative importance of TRH and TRbeta in feedback regulation of the HPT axis, we generated three groups of KO mice, each deficient in either or both protein(s). To establish that double KO mice were not expressing TRH, we measured hypothalamic TRH mRNA using in situ hybridization histochemistry (Fig. 1A). As expected, TRH mRNA was absent in TRH KO and double KO mice, whereas TRbeta KO mice demonstrated an increase in the TRH expression in the PVN when compared with WT animals (Fig. 1B). The latter result is consistent with a defect in negative T3 regulation of the TRH neuron as reported previously (20). Double KO mice were born with no gross anatomic or functional abnormalities and were viable through adulthood. Both male and female mice displayed normal fertility.


Figure 3
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  		FIGURE 3.
Analysis of the TSH bioactivity in WT, TRbeta KO, TRH KO, and double KO mice. A, cyclic AMP levels, TSH bioactivity expressed as a cAMP/TSH ratio, and serum T4/serum TSH are shown. Data were obtained from samples of sufficient volume to measure serum TSH, T4, and cAMP. B, histological analysis of thyroid glands. C, thyroid area and body weight. Thyroid area was determined from histological sections through the largest area of the thyroid gland. Data are shown as means ± S.E. *, p <0.01 or less. Five to six animals were evaluated in each group.

 
As previously reported, the absence of TRbeta results in a defect in negative feedback regulation of the HPT axis, resulting in higher TH levels in these mice. As shown in Fig. 2, total serum T3 and T4 levels were highest in the TRbeta KO mice, reaching statistical significance versus WT mice (15, 16, 27). Consistent with previous reports, TRH KO mice presented with decreased serum T4 levels when compared with WT animals (p <0.001). Interestingly, double KO mice displayed reduced T4 levels similar to that observed in TRH KO mice (p <0.001, Fig. 2). Serum T3 levels in both TRH KO and double KO were similar to WT mice, perhaps reflecting an increase in serum 5'-deiodinase activity in these animals. We also measured TH levels in animals with heterozygous genotypes generated from our breeding strategy (data not shown). TRH haploinsufficiency alone or combined with homozygous TRbeta deletion was insufficient to change basal thyroid hormone levels compared with WT and TRbeta KO animals, respectively. Furthermore, TRbeta haploinsufficiency alone or combined with homozygous TRH deletion was insufficient to change basal thyroid hormone levels compared with WT and TRH KO animals, respectively. Therefore we excluded these heterozygous genotypes from further study.

As expected, TRbeta KO animals showed resistance to TH and presented with elevated serum TSH levels (an 8-fold increase) when compared with all other groups (p <0.001, Fig. 2). TRH KO mice and double KO, in contrast, had only a 3-fold increase in serum TSH levels compared with WT animals. Because TRbeta was deleted in three of the four genotypes, we wanted to confirm that expression of the remaining TR{alpha}1 isoform was not altered in these animals. We found no significant differences in TR{alpha}1 mRNA levels in the pituitary of all genotypes studied (data not shown).

To determine serum TSH bioactivity in these animals, an in vitro cAMP assay was utilized. Fig. 3A, upper panel, demonstrates that serum from all three KO genotypes increased cAMP levels in this assay when compared with WT mice. TSH bioactivity (middle panel) was then determined by correcting for serum TSH levels (cAMP/TSH ratio). After this correction, TSH bioactivity was decreased in all KO groups when compared with the WT mice and was significantly decreased in TRH and double KO mice. Another measure of TSH bioactivity is the serum T4/TSH ratio (28).4 The T4/TSH ratio (Fig. 3A, lower panel)ofall KO groups showed a significantly decreased ratio compared with WT animals. This assay, however, underestimates TSH bioactivity when TSH values are markedly elevated due to the linear-log relationship between changes in T4 and TSH. A third measure of TSH bioactivity is a histological assessment of thyroid gland size (Fig. 3B). Thyroid glands were smaller in TRH and double KO mice (Fig. 3C, lower panel), consistent with a decrease in TSH bioactivity (based on two assays) in these mice versus WT mice. In contrast, these animals were of normal body weight (Fig. 3C, upper panel). Because of the younger age of our animals, we did not observe an increase in thyroid gland size in TRbeta KO mice, which is typically found after 6 months of age.


Figure 4
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  		FIGURE 4.
Effect of thyroid hormone deficiency and excess on regulation of the HPT axis. A, serum TSH levels were sequentially determined in WT and KO mice after a LoI/PTU diet + MMI water for 35 days followed by treatment with escalating L-T3 doses (0.2, 0.5, and 1 µg/100 g of body weight/day) for 7 days each. B, serum TSH at baseline, after LoI/PTU diet, and after the highest dose of L-T3 treatment. C and D, linear and log scales, respectively, of TSH-{alpha} and TSH-beta subunit mRNA levels in the anterior pituitary using a quantitative reverse transcription PCR analysis. Data were normalized for each mRNA level relative to the WT animals. Five to ten animals were evaluated in each group, and data are shown as means ± S.E. *, p <0.01 or less versus WT.

 
To investigate further the regulation of the HPT axis in these animals, all four groups were examined in both a thyroid hormone deficient and an excess state. After 35 days of a LoI/PTU diet supplemented with MMI in the water, all mice except the double KO animals presented with undetectable T4 levels (data not shown) and markedly elevated TSH levels (>10,000 milliunits/liter, Fig. 4A). In the double KO group, serum T4 levels were undetectable but serum TSH levels did not increase as much during hypothyroidism (<10% of hypothyroid WT levels, Fig. 4, A and B). L-T3 treatment was then administered by daily subcutaneous injections at three different concentrations. At the end of the low dose period, serum TSH decreased in all groups but the decline was more marked in the TRH KO mice. After the highest L-T3 dose, all groups had serum T3 levels of ~200 ng/dl, with no significant differences among the groups (data not shown). Serum TSH levels, however, were statistically significantly lower in the WT and TRH KO mice versus TRbeta and double KO mice (Fig. 4A, inset). Thus, L-T3 treatment reduced serum TSH in every group, but both the double KO mice and, as reported previously, the TRbeta KO mice demonstrated a resistance to L-T3 suppression at the highest dose (2, 27).

Like serum TSH, TSH subunit mRNA levels in double KO mice were not significantly elevated after PTU treatment (Fig. 4, C and D). TSH-{alpha} and -beta subunits mRNA responded to PTU treatment in all groups (except double KO animals). However, the magnitude of TSH-{alpha} subunit mRNA response was lower when compared with the TSH-beta subunit mRNA levels (Fig. 4, C and D, respectively).

To define the number of TSH-producing cells in the pituitary, TSH-beta immunopositive cells were quantitated in all groups after induction of hypothyroidism (Fig. 5). After 35 days of LoI/PTU + MMI treatment, the number of TSH-beta immunopositive cells, corrected for total cells in the field, was similar in WT and TRbeta KO mice (Fig. 5B, lower panel). In contrast, the number of TSH-beta immunopositive cells was somewhat lower in TRH KO mice and significantly lower in the double KO mice. Fewer TSH-beta immunopositive cells were observed throughout the anterior lobe of the double KO animals (p <0.01) when compared with WT mice, correlating with measurements of serum TSH levels at the end of the hypothyroid period (Fig. 5C).

We next evaluated pituitary TSH-beta immunostaining in hypothyroid animals after 10 days of treatment with a slow release TRH or placebo pellet (Fig. 6). After LoI/PTU + placebo treatment for 3 weeks, pituitary TSH-beta immunostaining (Fig. 6A) and serum TSH levels (data not shown) were similar to the results shown in Fig. 5 for WT and double KO mice. After TRH treatment, the number of detectable TSH-beta immunopositive cells significantly increased in double KO animals and was not different from WT animals (Fig. 6, A and B) or TRH KO and TRbeta KO mice (data not shown). Importantly, serum TSH levels were also similar after TRH treatment (TSH WT, 2943 ± 2427; TRbeta KO, 3709 ± 2654; TRH KO, 3045 ± 1693; DKO, 2923 ± 630 milliunits/liter).


Figure 5
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  		FIGURE 5.
Immunohistochemical analysis of the pituitary in WT and KO mice. A, immunohistochemical staining for TSH-beta in the pituitary after 35 days of LoI/PTU-MMI treatment. Positive TSH-beta cells are white in the low magnification images and dark in the high magnification images. B, the number of immunoreactive (IR) TSH-beta cells was normalized to the total number of cells. Five animals were evaluated in each group. C, serum TSH after LoI/PTU-MMI treatment. *, p <0.01 or less versus WT.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The physiological set point for TSH synthesis and release is determined by a balance between a positive input from hypothalamic TRH and a strong negative influence by thyroid hormone. Together these opposing influences determine the amount of TSH synthesized and secreted. It has also been demonstrated in TR KO animals that TRbeta2 is the dominant isoform mediating T3 negative regulation of TSH subunit gene expression in the pituitary and that TRbeta2 is the main isoform regulating TRH gene expression in the hypothalamus (16, 20). The importance of TRbeta2 in regulating the HPT axis may reflect a higher expression level of this isoform in tissues regulating the HPT axis, because it has been shown that somatic gene transfer of either TRbeta1 or TRbeta2 rescues the hypothalamic defect in TRH negative regulation in TRbeta KO animals (29). In contrast, TR{alpha}1 deletion alone or in combination with {alpha}2 causes a mild central hypothyroidism, perhaps because of a regulatory or developmental defect in the HPT axis (13, 14, 30).

Hypothyroidism is necessary but not sufficient to up-regulate the HPT axis. This was suggested by a study of patients with central hypothyroidism caused by hypothalamic dysfunction and definitively shown in mice lacking TRH (26, 31). Hypothalamic TRH deficiency in humans and rodents has been shown to decrease TSH bioactivity by changing its glycosylation pattern (31, 32). In humans and rodents lacking TRH, decreased TSH bioactivity was demonstrated by an increase in serum TSH levels relative to WT animals associated with a reduction in serum T4 levels. The reduction in TH levels and change in TSH glycosylation were then corrected after exogenous administration of TRH to TRH-deficient animals (26, 31). In addition to an effect on TSH bioactivity, TRH deficiency is also thought to reduce the number of pituitary thyrotrophs in adult mice (25).


Figure 6
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  		FIGURE 6.
Immunohistochemical analysis of the pituitary in WT and double KO mice. A, immunohistochemical staining for TSH-beta in the pituitary after 3 weeks of LoI/PTU-MMI treated with or without TRH. B, the number of immunopositive TSH-beta cells was normalized by total number of cells. Five animals were evaluated in each group.

 
The relative importance, however, of these positive and negative inputs in the central axis has yet to be determined in vivo. By deleting either TRbeta, TRH, or both in mice, we sought to address this question. As previously reported, TRbeta KO animals demonstrated significant increases in TH and TSH levels at 16 weeks of age, confirming resistance to TH. Also as reported, TRH KO mice have reduced T4 levels associated with a 2–3-fold increase in serum TSH levels. Interestingly, double KO mice were indistinguishable from TRH KO animals.

Our data in TRH and double KO mice demonstrate lower serum T4 levels associated with higher serum TSH values, suggesting a decrease in the TSH bioactivity. TSH bioactivity was measured directly by determining cAMP generation in an in vitro TSH bioassay. Although serum TSH levels were higher in all KO groups, cAMP/TSH ratios (a measure of TSH bioactivity) were decreased when compared with WT animals (Fig. 3A). This finding most likely illustrates the critical role that TRH plays in TSH glycosylation in the anterior pituitary, as previously reported (31–33). We did not observe goiter in TRbeta KO animals even though their absolute TSH level was elevated (Fig. 3, B and C). This may be because of the age of the mice used in our study; we find that goiter in TRbeta KO animals is age dependent. In contrast, the thyroid gland was smaller in TRH and double KO mice, suggesting that the increased serum TSH levels in these mice could not compensate for a reduction in serum TSH bioactivity.

We next studied negative regulation of the central axis by T3 after animals were rendered hypothyroid. As previously reported (27), TRbeta KO animals were less responsive to T3, such that at the highest T3 dose TSH was still markedly elevated (Fig. 4A, inset). Double KO mice behaved as TRbeta KO animals even though TSH levels were much lower at the beginning of T3 treatment (see below). In contrast, the responsiveness of the thyrotroph to exogenous T3 was increased in TRH KO mice; TSH values returned rapidly with the smallest T3 dose. This result suggested that the thyrotroph might be more sensitive to T3 negative feedback without hypothalamic TRH input. The most unexpected result in this study was that thyrotrophs from double KO mice failed to respond to hypothyroidism. This experiment was designed to ensure that all the groups became equally hypothyroid before administration of T3 (T4 was undetectable in all groups treated with LoI/PTU and MMI). Serum TSH levels were at least 50-fold lower and TSH subunits mRNA levels at least 2-fold lower in double KO mice during the hypothyroid phase of the experiment. Given that we observed appropriately increased serum TSH levels in WT, TRH KO, and TRbeta KO mice, we concluded that the presence of both TRbeta and TRH is necessary for a normal thyrotroph response during hypothyroidism, suggesting that unliganded TRbeta stimulates TSH subunit gene expression (Fig. 4). Support for these findings can be found in previous studies of primary thyrotroph cell cultures that demonstrated that either TRH or the absence of TH was sufficient to mediate an increase in TSH subunit gene transcription (2, 34).

To explore further the mechanism for the decreased serum TSH in double KO mice, we measured the number of TSH-beta immunopositive cells in pituitary sections. Compared with WT animals, the number of TSH-beta immunopositive cells was similar in TRbeta KO mice, somewhat decreased (but not significantly) in TRH KO mice, and markedly decreased in double KO animals during hypothyroidism. Because the number of detectable TSH-beta immunopositive cells was decreased in the double KO during induced hypothyroidism (Fig. 5, A and B), these data could suggest that the combination of TRH and TH (via TRbeta) are required for thyrotroph cell development and/or maintenance.

To confirm this hypothesis, we evaluated the response of KO animals to TRH stimulation (Fig. 6). Slow release TRH or placebo pellets were implanted in hypothyroid animals. After this treatment, no significant differences in either TSH-beta immunopositive cells or serum TSH levels were found among the groups, indicating that the defect in double KO mice was corrected after TRH treatment. These results indicate that the decreased serum TSH values observed in the double KO animals during hypothyroidism are due to decreased TSH synthesis and not due to a reduction in thyrotroph cell number.

Others have shown that TRH is not physiologically required for the proliferation or differentiation of embryonic thyrotrophs. After birth, however, the number of TSH-beta immunopositive cells decreases, showing the importance of TRH in maintenance of normal postnatal functions of the pituitary thyrotrophs (25). Our results show that TRH KO mice respond normally or nearly normally to hypothyroidism but that double KO mice display a significantly impaired response. Serum TSH failed both to increase normally after hypothyroidism and to suppress normally after T3 administration. These data suggest a previously unrecognized interaction between TRH and TH signaling pathways in mediating the hypothyroid TSH response. We can also speculate that TRH signaling may enhance stimulation by the unliganded TRbeta by an unknown cross-talk mechanism.

In conclusion, TRH is critical for normal regulation of the HPT axis. TRH absence causes central hypothyroidism in mice due to the synthesis of biologically less active TSH. When challenged with primary hypothyroidism, however, the central axis is also unable to respond normally in mice lacking TRH, even when negative feedback is also disrupted (double KO mice). This defect in double KO mice reflects a marked decrease in TSH synthesis, which was reversed by chronic TRH stimulation. Although hypothyroidism is known to markedly increase TSH synthesis at a transcriptional level, these results indicate an unexpected, dominant role for TRH in regulating the HPT axis in the basal and hypothyroid state.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK49126 and DK53036 (to F. E. W.) and Grant DK20595 from the Diabetes Research and Training Center at the University of Chicago. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Division of Metabolism, Depts. of Pediatrics and Medicine, Johns Hopkins Medical Institutes, Baltimore, MD 21287. E-mail: fwondisford{at}jhmi.edu.

3 The abbreviations used are: TH, thyroid hormone; 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, thyroxine; TR, thyroid hormone receptor; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; WT, wild-type. Back

4 S. Refetoff, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Sally Hall for technical assistance with the in situ hybridization histochemistry.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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