help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0236
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moeller, L. C.
Right arrow Articles by Weiss, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moeller, L. C.
Right arrow Articles by Weiss, R. E.
Right arrowPubmed/NCBI databases
*Gene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*LEVOTHYROXINE
Endocrinology Vol. 148, No. 10 4727-4733
Copyright © 2007 by The Endocrine Society

Pituitary-Thyroid Setpoint and Thyrotropin Receptor Expression in Consomic Rats

Lars C. Moeller, Manuela Alonso, XiaoHui Liao, Vance Broach, Alexandra Dumitrescu, Jacqueline Van Sande, Lucia Montanelli, Stephen Skjei, Charles Goodwin, Helmut Grasberger, Samuel Refetoff and Roy E. Weiss

Departments of Medicine (L.C.M., M.A., X.L., V.B., A.D., S.S., C.G., H.G., S.R., R.E.W.) and Pediatrics (R.E.W., S.R.), Committees on Genetics and Molecular Medicine (S.R., R.E.W.), The University of Chicago, Chicago, Illinois 60637; and Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (J.V.S., L.M.), University of Brussels, School of Medicine, Campus Erasmus, B 1070 Brussels, Belgium

Address all correspondence and requests for reprints to: Roy E. Weiss, M.D., Ph.D., The University of Chicago, MC 3090, Chicago, Illinois 60637. E-mail: rweiss{at}medicine.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The genetic basis for differences in TSH sensitivity between two rat strains was examined using consomic rats generated from original strains salt-sensitive Dahl (SS) (TSH 1.8 ± 0.1 ng/ml; free T4 index 4.9 ± 0.4) and Brown Norwegian (BN) (TSH 5.5 ± 0.6 ng/ml, P < 0.05; free T4 index 4.3 ± 0.1, P not significant). Consomic rats SSBN6 [BN chromosome (CH) 6 placed in SS rat] and SSBN2 (BN CH 2 placed in SS rat) have TSH concentrations intermediate between pure SS and BN strains (2.9 ± 0.3 and 3.1 ± 0.3 ng/ml, respectively; P < 0.05). Candidate genes on rat CH 2 included TSH ß-subunit and on CH 6 the TSH receptor (TSHR). TSH from sera of BN, SS, SSBN6, and SSBN2 strains had similar in vitro bioactivity suggesting that the cause for the variable TSH concentrations was not due to an altered TSH. Physiological response to TSH was measured by changes in serum T4 concentrations upon administration of bovine TSH (bTSH). Rat strain SS had a greater T4 response to bTSH than BN (change in T4, 1.3 ± 0.1 vs. 0.4 ± 0.1 µg/dl, P < 0.005), suggesting reduced thyrocyte sensitivity to TSH in BN. Sequencing of the TSHR coding region revealed an amino acid difference in BN (Q46R). This substitution is unlikely to contribute to the strain difference in serum TSH because both TSHR variants were equally expressed at the cell surface of transfected cells and responsive to bTSH. Given similar TSH activity and similar TSHR structure, TSHR mRNA expression in thyroid tissue was quantitated by real-time PCR. BN had 54 ± 5% the total TSHR expression compared to SS (100 ± 7%, P < 0.0001), when corrected for GAPDH expression, a difference confirmed at the protein level. Therefore, the higher TSH level in the BN strain appears to reflect an adjustment of the feedback loop to reduced thyrocyte sensitivity to TSH secondary to reduced TSHR expression. These strains of rat provide a model to study the cis- and trans-acting factors underlying the difference in TSHR expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REGULATION OF SERUM TH levels is dependent on the hypothalamic and pituitary control of TH release. In mammals, a narrow range of TH concentrations is maintained by a broad normal range of TSH, indicating variable sensitivity to TSH. There is considerable evidence to suggest that TSH sensitivity is genetically controlled in part; however, the genes involved have yet to be identified (1, 2, 3). We have previously reported that thyroid hormone levels, specifically TSH values, vary in different strains of mice with much less variation in serum T4 levels (4). Serum TSH concentrations also differ among Wistar and Wistar-Kyoto rat strains (5).

Candidate genes involved in the regulation of TH levels are those for TSH, the TSH receptor (TSHR), or other factors that regulate their expression, metabolism, and action. TSH, a pituitary glycoprotein, is composed of a common {alpha}-subunit and a TSH-specific ß-subunit coded by genes located on human chromosomes (CH) 6 and 1, respectively (6). The two subunits are noncovalently linked and are species specific. A model of TSH has been developed where the two subunits are stabilized by a unique segment of the ß-subunit termed the "seat belt" because it wraps around the {alpha}-subunit long loop (7, 8). The seat belt is crucial for the heterodimer stability and function of the TSH (9, 10). The folding at this point allows for glycosylation of the molecule that is important for its function (11). Various clinical states can alter the degree of glycosylation of TSH including central hypothyroidism, euthyroid sick syndrome, aging, chronic uremia, and resistance to thyroid hormone (12). In addition, TRH itself can enhance the biological activity of TSH by modifying its glycosylation pattern (13, 14, 15). Mutations in TSHß usually result in a molecule that is quickly degraded and low in serum concentration causing marked hypothyroidism (16, 17, 18, 19, 20).

The TSHR is a member of the class of glycoprotein hormone receptors that constitute a subfamily of G protein-coupled receptors with a characteristic modular structure. The carboxyl-terminal portion is made up of a rhodopsin-like portion with seven transmembrane {alpha}-helices and is responsible for transduction of the intracellular signal, mainly through Gs {alpha} (21, 22, 23, 24, 25). There are also naturally occurring mutations of the TSHR that result in an inactivation of the receptor and subsequent resistance to the action of TSH (26, 27).

The genetic basis for the variation in the set points of thyroid/pituitary feedback regulation in the human population is unknown. We report on a consomic rat model to study this phenomenon. Brown Norwegian (BN) rats have higher serum TSH levels than salt-sensitive Dahl (SS) rats despite similar free T4 levels. In consomic rats, an entire CH from one strain of rat is replaced in another strain, allowing analysis of the influence of genes contained on one CH in another genetic background. We describe that transfer of either BN CH 2 or 6 in an SS background (SSBN2, SSBN6) partially transfers the phenotype of high TSH to these consomic strains. We demonstrate that reduced TSHR expression is linked to the difference in serum TSH levels mechanistically in the presence of the same TH concentration. This rat model will allow mapping of cis- and trans-acting factors underlying the variability in TSHR expression and could provide a paradigm underlying differences in thyroid/pituitary feedback regulation in the human population.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All rats used were male and obtained from the Medical College of Wisconsin (MCW) at Milwaukee through the PhysGen (Programs for Genomic Applications) project. Parental strains, SS (SS/JrHsd/Mcwi), FHH (Fawn-Hooded Hypertensive; FHH/EurMcwi), and BN (BN/NHsdMcwi) were bred at Charles River Laboratories (Wilmington, MA). Rats were kept at the barrier facility at MCW until shortly before the study, at which time they were moved to a holding room outside the barrier. The animals were fed 0.4% NaCl Teklab 3075S chow. Rats were weaned at 3 wk of age at the MCW and were studied shortly thereafter. Some rats were transported to the University of Chicago for additional studies described. All animal experiments were performed at The University of Chicago according to protocols approved by the Institutional Animal Care and Use Committee.

Generation of the inbred "consomic" rats was under the auspices of the Genomics Component of PhysGen at the MCW. In consomic rats, one CH from one parental strain replaces the CH of another "background" strain through breeding the rats and selection of progeny having genetic markers. In this study, we examined rats with a SS genomic background that had their second and sixth CH replaced with that of BN rats. Thyroid function tests were determined on a total of 329 rats including the SS, BN, and FHH rats as well as the 20 consomic SSBN strains. For the additional experiments, described below, a total of nine BN, nine SS, nine SSBN2 (Consomic Chromosome 2 rats), and seven SSBN6 (Consomic Chromosome 6 rats) were studied.

TSH stimulation test
Thyroid response to TSH was determined after ip injection of bovine TSH (bTSH; Sigma, St. Louis, MO). Rats were pretreated for 4 d with 5 µg L-T3 per 100 g body weight per day ip to suppress endogenous TSH production and reduce serum T4 concentration. Four different doses of TSH were administered on the morning of the fifth day. Blood was obtained before and 30 min after the injection. The dose of TSH was adjusted based on a TSH stimulation test developed in our laboratory for use in mice (28).

TSHR and TSH ß-subunit sequencing
Sequencing of the TSHR and TSH ß-subunit was performed for the four rat strains studied. mRNA was extracted from one lobe of the thyroid gland of the rats studied. Two micrograms of extracted RNA were reverse transcribed to cDNA using random hexamers with the First-Strand Synthesis Superscript Kit (Life Technologies, Rockville, MD) following the provided protocol. All exons for TSHR and TSHß were amplified by PCR (sequences of primers and reaction conditions available upon request). Reactions were analyzed by agarose gel electrophoresis, purified and subjected to DNA cycle sequencing (Big Dye; Applied Biosystems, Foster City, CA).

Measurements of T4 and TSH by RIA and TSH bioactivity
Serum TSH was measured in 50 µl of serum using a sensitive, heterologous, disequilibrium double antibody precipitation RIA as previously described (4). Samples containing more than 5 ng/ml TSH were 5- and 50-fold diluted with a TSH-deficient rat serum. Serum T4 concentrations were measured by a antibody-coated solid phase RIA (Diagnostic Products, Los Angeles, CA) using 25 µl of serum. The sensitivity of this assay was 0.2 µg/dl. Free T4 was estimated from the resin T4 uptake test and the total T4 values and expressed as the free T4 index (FT4I) index.

Bioactivity of serum TSH was determined by measurement of cAMP generation following the addition of serum to the medium of cultured Chinese hamster ovary (CHO) cells stably transfected with a TSHR cDNA. The method was essentially that described for the measurement of TSH bioactivity in mouse serum (4). The only difference was the use of rat rather than mouse TSH-deficient serum. The former (rTSH-0) was produced by treatment of rats with drinking water containing 5 µg/ml L-T4 (~50 µg/150 g rat · d) for 2 wk. Rats were exsanguinated under anesthesia, and sera were collected. Purified rat TSH (NIDDK-rTSH-RP-3, provided by the National Hormone and Pituitary Program) was added in different concentration to the rTSH-0 serum pool for the construction of a standard curve. Results of cAMP measurements, described below, were correlated to those of the TSH immunoassay by linear regression. To calculate the intrinsic TSH bioactivity, i.e. the ratio of biological activity to the amount of immunoreactive TSH, the cAMP results were corrected for TSH-independent cAMP generation by subtracting the y-intercept of the (uncorrected) standard regression line. The sensitivity of the bioassay is 0.02 ng rat TSH per milliliter. This produces, on the average, a 5-fold increase in the basal cAMP level. The interassay variability for the range of TSH values measured was 5%.

In vitro expression of rat TSHR codon 46 variants
Plasmids encoding the TSHR variants were constructed by site-directed mutagenesis using PCR amplifications as described previously (29). An amplified fragment containing the variant, confirmed by sequencing, was cloned after digestion with appropriate restriction enzymes into the pSVL (simian virus 40 late promoter) expression vector (Amersham Pharmacia Biotech, Freiburg, Germany) containing the wild-type TSHR (30). COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone. Cells were seeded at a density of 350,000 cells per cubic centimeter dish. One day later, they were transfected (500 ng DNA per dish) by the diethylaminoethyl-dextran method followed by a dimethylsulfoxide shock (31). Two days after transfection, cells were used for fluorescence-activated cell sorting (FACS) analysis and cAMP studies. Duplicate dishes were used for each assay. Each experiment was repeated at least three times. Cells transfected with pSVL alone were always ran as controls.

FACS analysis and cAMP determination
Surface expression of the variant rat TSHR transiently transfected into COS7 cells was determined as described previously in studies of human TSHR variants (29). After transfection, COS cells were detached from the dishes with PBS containing 5 mM each of EDTA and EGTA, transferred into tubes, and centrifuged at 500 x g for 3 min at 4 C, and the supernatant was removed. Pelleted cells were incubated for 30 min with monoclonal antibody to TSHR (IRI-SAB2) diluted in PBS with 0.1% BSA. IRI-SAB2 was produced by genetic immunization with the wild-type TSHR cDNA and recognizes only the native receptor as expressed at the cell surface (32). Cells were then washed with 4 ml PBS-1% BSA, centrifuged, and incubated for 30 min on ice and in the dark with fluorescein-conjugated {gamma}-chain-specific goat antimouse IgG (Sigma). Propidium iodide (10 µg/ml) was used for the detection of damaged cells that were excluded from the analysis. Cells were washed once again and resuspended in 250 µl PBS-0.1% BSA. The fluorescence of 5000 cells per tube was assayed by FACScan Flow Cytofluorometer (Becton Dickinson, San Jose, CA).

For cAMP determination, cells were washed with Krebs-Ringer-HEPES buffer (KRH isotonic, pH 7.4). After a preincubation in KRH at 37 C for 30 min, cells were incubated in the same buffer supplemented with 25 µM Rolipram (a cAMP phosphodiesterase inhibitor; a gift from the laboratory of J. Logeais, Paris, France), in the absence or presence of various bTSH concentrations (Sigma). One hour later, the medium was removed and 0.1 M HCl was added to the cells. The cellular extracts were dried overnight in a vacuum concentrator (Savant, Holbrook, NY) and intracellular cAMP was determined exactly as described previously (33). Basal cAMP was normalized to cell surface expression for each of the constructs. To this end, specific cAMP accumulation (cAMP of receptor transfected cells minus cAMP of the pSVL-transfected cells) is divided by the specific FACS value (fluorescence of receptor-transfected cells minus fluorescence of pSVL-transfected cells), which can be summarized as: specific basal activity [cAMP(receptor) – cAMP(pSVL)]/[FACS(receptor) – FACS(pSVL)]. The values are then expressed as the percentage of specific basal activity of the wild-type TSHR.

Determination of SS and BN TSHR promoter activity
FRTL-5 cells were grown in Ham’s F12K medium containing 2 mM [;scap];l[;r];-glutamine (ATCC, Manassas, VA) adjusted to contain 1.5 g/liter sodium bicarbonate and supplemented with 5% fetal bovine serum and a six-hormone mixture (6H) comprising bTSH (10 mU/ml), insulin (10 µg/ml), hydrocortisone (10 nM), transferrin (5 µg/ml), somatostatin (10 ng/ml), and glycyl-L-histidyl-L-lysine acetate (10 ng/ml) (all from Sigma). Equal number of cells were seeded into six-well plates before transfection. The promoter region of the TSHR was amplified from gDNA of SS and BN rats using primers with Mlul and Xho1 restriction sites and directionally cloned upstream of the firefly luciferase reporter gene in plasmid PGL3 (Promega, Madison, WI). FRTL-5 cells were transfected for 9 h in 2% calf serum/OptiMEM-1 (Invitrogen, Carlsbad, CA) with 0.1–1.0 µg reporter vector and 0.1 µg pRL-Tk (Promega) using Lipofectamine 2000 (Invitrogen). Subsequently, cells were cultured without or with bTSH in the medium. Thirty-six hours posttransfection, cells were harvested and analyzed sequentially for firefly and Renilla luciferase activities (Dual-Luciferase Reporter Assay System; Promega). Firefly luciferase activity was normalized for the corresponding Renilla activity to correct for differences in transfection efficiency.

Real-time PCR
Reactions for the quantification of TSH receptor mRNAs were performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using SYBR Green I as detector dye in. The reaction mixtures contained 25 µl QuantiTect SYBR Green PCR Kit (QIAGEN Inc., Valencia, CA), 0.3 µmol/liter of each primer, 10 ng template cDNA, and RNase-free water to a final volume of 50 µl, and were performed in 96-well plates. Cycle conditions were 50 C for 2 min, 95 C for 10 min followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. The primers spanned exons 8 and 9 of the TSH receptor and were: forward 5'-TGG AGG AGT ATA CAG TGG ACC CA-3', and reverse 5'-CTT TGA GGT GCT CCA GGC C-3'.

The expression of the gene was calculated relative to the SS parental strain and normalized against the expression of GADPH mRNA using the 2–{Delta}{Delta}CT method (34).

Measurement of TSH-R protein expression
Thyroids were uniformly dissected by the same investigator. First, the trachea with thyroids and sternohyoid muscles was removed from the body. Then, under a dissecting microscope, the muscles were removed completely and the intact thyroid lobes lifted off the trachea. The thyroids were then shock frozen on dry ice and weighed on a mechanical balance before storage at –80 C. Thyroid glands dissected from two SS and two BN rats, were homogenized in 50 mM Tris-HCl pH 7.4, 150 mM NaCl2, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin, and then sonicated as previously described (35). Samples were centrifuged at 10,000 rpm at 4 C for 10 min, and supernatant proteins were quantified by Bradford’s method using BSA as a standard (Bio-Rad, Hercules, CA). Samples containing 50 µg protein were separated on a 10% SDS-PAGE gel before transfer to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). Membranes were incubated in nonfat dried milk (20%) in Tween-PBS (0.1%) at room temperature for 2 h, followed by incubation in nonfat dried milk (5%) in Tween-PBS (0.1%) containing the TSHr antibody 3G4 (35) 1:100 at 4 C for 16 h. Membranes were washed in Tween-PBS (0.1%) and incubated with goat antimouse horseradish peroxidase 1:2000 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature before washing in Tween-PBS (0.1%). Bands were visualized using luminol reagent (Santa Cruz Biotechnology). To control for loading, the membrane was stripped and probed with anti-fibronectin (Sigma) at 1:2000 dilution and developed again.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thyroid function tests of SS and BN rats
We found evidence for a significant difference in TSH sensitivity between SS [TSH 1.8 ± 0.1 ng/ml (mean ± SD); FT4I 4.9 ± 0.4 µg/dl] and BN (TSH 5.5 ± 0.6 ng/ml P < 0.05; FT4I 4.3 ± 0.1 µg/dl, not significant) rat strains (Table 1Go). To identify which CH harbor major effect quantitative trait loci for TSH sensitivity, we examined SSBN consomic strains for all 20 autosomes and for the X and Y CHs. SSBN6 and SSBN2 had TSH values intermediate between SS and BN (3.1 ± 0.3 and 2.9 ± 0.3 ng/ml, respectively) (Table 1Go). The fact that the TSH values are intermediate suggest that genes from both CH may be responsible for a genetically determined set-point. We surveyed the annotated rat genome (36) for potential candidate genes. The most relevant on CH 2 appears to be Tshb (encoding the TSH ß-subunit), although on CH 6 several plausible candidate genes are found: Tshr, Titf1 (thyroid transcription factor 1), Tpo (thyroid peroxidase), and Dio2 (iodothyronine deiodinase, type 2) and Dio3 (iodothyronine deiodinase, type 3).


View this table:
[in this window]
[in a new window]

  		TABLE 1. TFTs in SS, FHH, BN and consomic rats (mean ± SD)

 
TSH sequence and bioactivity
There were no differences observed in the exon and flanking intron sequence of TSHß from SS and BN rats. In addition, TSH bioactivity was measured in serum of BN, SS, SSBN6, and SSBN2 rats, and no differences, relative to immunoactivity, were found. (Fig. 1Go).


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 1. TSH bioactivity. Correlation of rat TSH bioactivity (cAMP generation) to immunoreactivity in serum from the original rat strains (BN and SS) and consomic (SSBN6 and SSBN2). The regression line is plotted for results generated from purified rat TSH reference standard.

 
In vivo measurement of TSH action on thyroid follicular cells
Because TSH from the two strains was apparently normal in structure and intrinsic bioactivity, we investigated the response of the thyroid follicular cells to exogenous TSH. Rats were given bTSH after 4 d of suppression with T3, and changes in endogenous serum T4 were measured. Baseline T4 was suppressed less than 0.2 µg/dl in all rats. SS had a greater increase of T4 in response to bTSH than BN (T4, 1.3 ± 0.1 vs. 0.4 ± 0.1 µg/dl, P < 0.005) at 10 mU bTSH per 100 g body weight, indicating reduced thyroid follicular cell sensitivity to TSH in BN rats (Fig. 2Go). Similar differences were seen with the other three doses of bTSH used. Thyroid gland size was not different between SS and BN rats (32 ± 2 vs. 30 ± 2 mg thyroid per 100 g body weight, respectively; not significant). A similar thyroid size of BN and SS rats despite higher TSH concentrations with normal bioactivity in the BN rats supports the finding of decreased thyroid sensitivity to TSH in this strain.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 2. In vivo response to exogenous bTSH (TSH stimulation test). Different doses of bTSH were given ip to groups of five to nine rats pretreated with L-T3. Serum T4 was measured before and after 30 min. Results are mean ± SD. Statistical significance is indicated. Note that baseline T4 in all rats was less than 0.2 µg/dl.

 
TSH receptor sequence and in vitro function and expression
Sequencing the complete Tshr gene coding region of the two rat strains revealed a nucleotide difference predicting a single amino acid difference at codon 46 [46Q (CAA) in SS, 46R (CGA) in BN]. Both TSHR variants were expressed in COS cells and assessed for TSH-stimulated cAMP production (Fig. 3Go). No differences in basal or maximum stimulated cAMP generation, or the EC50 of the TSH response curves were found for the two variant rat TSHR. Flow cytometry confirmed that both variants were expressed at the same level on the surface of transfected cells (Fig. 4Go). These results indicate that the variant at codon 46 of the Tshr gene is unlikely to contribute to the difference in response to TSH between SS and BN rats.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 3. In vitro function of the TSHR variants [46Q (CAA) in SS, 46R (CGA) in BN]. Effect of bTSH on cAMP accumulation in COS-7 cells transfected with the human TSHR expression vectors.

 

Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		FIG. 4. In vitro expression of the TSHR variants. Cell surface expression of Q46 (SS) and R46 (BN) variant rat TSHR in transfected COS-7 cells determined by flow cytometry.

 
TSHR mRNA levels and protein content in rat thyroid gland
Because the TSHRs of SS and BN strains function similarly in vitro, whereas the thyroids in the BN rats are less responsive to bTSH, we measured the level of expression of the Tshr gene in the thyroid glands of these rats. BN expressed 54 ± 5% of the Tshr mRNA compared with the SS rat (100 ± 7%, P < 0.0001 when corrected with GAPDH) (Fig. 5Go). This indicated that a reduction in Tshr expression could be the cause for the reduced response to exogenous TSH in the BN rats and hence their higher serum TSH concentration. The thyroid glands of BN rats had also a decreased amount of the TSHR protein analyzed by Western blotting (Fig. 6Go).


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 5. TSH receptor expression in the SS and BN rats. Quantitative PCR performed on mRNA extracted from SS and BN thyroid.

 

Figure 6
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 6. Western blot. SS and BN thyroid glands (two each and pooled) were extracted and protein was resolved on an SDS polyacrylamide gel and transferred to a membrane. TSHR was detected with the TSHR antibody 3G4 (see Materials and Methods). Protein was normalized with fibronectin.

 
Analysis of TSHR promoter activity in SS and BN strains
To evaluate whether strain differences in the Tshr gene promoter region could explain the differences in TSHR mRNA expression level, the region encompassing 3000 bp upstream of the translation start was sequenced in both strains. Fifteen single nucleotide substitutions and one dinucleotide deletion were found (Table 2Go). One of these differences were close to the TITF1 consensus binding sites and one was in a GABPB1 binding site. In vitro expression of the promoter region in FRTL5 cells showed no differences in the activities of the SS- and BN-specific promoters to drive reporter gene expression, both at baseline and after bTSH stimulation (Fig. 7Go). Although our results do not exclude a possible role of regions even further upstream of the transcription start site, it appears that differences in the promoter are unlikely to contribute to the reduced Tshr gene expression in the BN rats.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Strain sequence differences in the Tshr promoter region

 

Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 7. In vitro measurement of promoter activity in FRTL5 cells. The activities of SS- and BN-specific promoters to drive reporter gene expression were measured after bTSH stimulation across a wide range of concentrations. Promoter activity is given in relative light units (RLU) and Firefly luciferase activity was normalized for the corresponding Renilla activity to correct for differences in transfection efficiency.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TSH concentration differences in the presence of similar serum T4 levels reflect a difference in sensitivity of the thyrocyte and downstream pathways regulated by the TSH receptor. On the other hand, differences in T4 levels with similar serum concentrations of TSH reflect differences in sensitivity of the thyrotroph to TH usually involving its receptor. The combined regulation of sensitivity to feedback by both the thyrotroph and thyrocyte contribute to a genetically determined "set-point" at the hypothalamic-pituitary-thyroid axis. The interindividual variation of serum TSH level is much greater than that of the TH level, suggesting a more complex regulation at the thyrocyte level to explain the differences in these rat strains.

In humans, several lines of evidence suggest genetic regulation of the set-point: twin studies, genetic association studies, and familial studies. In a study of male twins, there was significantly less variability of TH and TSH than in unrelated subjects (3). Seventy-two percent of the variation in plasma free T4 concentrations was affected by familial factors in monozygotic brother pairs and 50% was affected in dizygotic twin pairs (P < 0.03). In another study, Peeters et al. (1) have shown a significant association of the TSHR D727E allele with lower plasma TSH levels. Measurement of TH concentrations in members of a large kindred demonstrated that TH levels were more similar in first degree relatives than in relatives by marriage (37). Finally the description of subjects with autosomal dominant resistance to TSH suggested an inherited syndrome with altered TSHR activity responsible for variable degrees of serum TSH (26, 38, 39). However, although there is evidence to suggest that the set-point of the TH axis is genetically controlled, the actual genes involved are unknown.

The inbred Wistar-Kyoto (WKY) rat strain that has a higher serum TSH with similar T4 values than their out-bred progenitor strain is another example of differences in thyroid set-point within a species. In the current study, we chose to evaluate the SS and BN rat strains as they also had significant differences in serum TSH and in addition, consomic strains were available for all CH. Consomics for CH 2 and 6 had serum TSH concentrations intermediate between SS and BN, suggesting that genes on both CH modulate the phenotype. Using quantitative trait locus analysis for the Wistar rats, a locus identified on CH 6 accounted for over 8% of the variance in serum TSH and had a LOD score of 11.7; however, the cause of the difference was not determined (5). Weaker LOD scores were found on CH 1 and 2 (LOD = 2.3) (5). Because our current studies also indicated CH 6, we evaluated in detail the Tshr genes of SS and BN strains. BN strains had a lower response to exogenous bTSH as measured by the increase of serum T4 over baseline, compatible with a relative resistance of BN strains to respond to TSH. A mechanistic link to the relative resistance to TSH is likely provided by the reduced expression of Tshr gene in the BN rats as determined by quantitative PCR and by Western blotting. We found sequence differences in the coding and promoter regions of the Tshr, but according to the results of in vitro studies these appear not to contribute to higher serum TSH level in the BN strain.

Taken together, these data indicate that there is a functional decrease in the action of TSHR that is not due to the single amino acid difference nor due to changes in the promoter sequence. We conclude that the higher TSH level in the BN strain can be explained by reduced TSHR expression and reflects an adjustment of the feedback loop to reduced thyroid sensitivity to TSH. These results show that variability in TSHR expression can explain the variation of serum TSH levels. Additional studies are necessary to determine the mechanism of altered Tshr gene expression and to determine the genes on CH 2 and other genes on CH 6 that influence thyrotroph sensitivity.


   Acknowledgments
 
We thank Drs. S. Costagliola and G. Vassart for the provision of antibodies to TSHR.


   Footnotes
 
This work was supported in part by Grants RR18372, DK17050, and DK20595 from the National Institutes of Health, The Seymour Abrams Thyroid Research Fund, and the Morris Esformes Endowment. Publication cost was defrayed by Provell Pharmaceuticals, LLC, Honeybrook, PA. V.B. was a recipient of an undergraduate student summer fellowship from The Endocrine Society. L.C.M. was a recipient of a grant from the Deutsche Forschungsgemeinschaft, DFG (Mo 1018/1-1). The PhysGen (Physiogenomics of Stressors in Derived Consomic Rats) program at the Medical College of Wisconsin at Milwaukee is funded by the National Heart, Lung and Blood Institute, U01 HL66579.

Present address for L.C.M.: Division of Endocrinology, Department of Medicine, University Hospital of Essen Medical School, 45122 Essen, Germany.

Present address for L.M.: Dipartimento di Endocrinologia e Malattie Metaboliche Via Paradisa, 2 (zona Cisanello) 56124, Pisa, Italy.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online July 19, 2007

Abbreviations: BN, Brown Norwegian; bTSH, bovine TSH; CH, chromosome; FACS, fluorescence-activated cell sorting; FHH, fawn hooded hypertensive; FT4I, free T4 index; pSVL, plasmid with the simian virus 40 late promoter; SS, salt-sensitive Dahl; TSHR, TSH receptor.

Received February 20, 2007.

Accepted for publication July 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Peeters RP, van Toor H, Klootwijk W, de Rijke YB, Kuiper GG, Uitterlinden AG, Visser TJ 2003 Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and iodothyronine levels in healthy subjects. J Clin Endocrinol Metab 88:2880–2888[Abstract/Free Full Text]
  2. Samollow PB, Perez G, Kammerer CM, Finegold D, Zwartjes PW, Havill LM, Comuzzie AG, Mahaney MC, Goring HH, Blangero J, Foley TP, Barmada MM 2004 Genetic and environmental influences on thyroid hormone variation in Mexican Americans. J Clin Endocrinol Metab 89:3276–3284[Abstract/Free Full Text]
  3. Meikle AW, Stringham JD, Woodward MG, Nelson JC 1988 Hereditary and environmental influences on the variation of thyroid hormones in normal male twins. J Clin Endocrinol Metab 66:588–592[Abstract/Free Full Text]
  4. Pohlenz J, Maqueem A, Cua K, Weiss RE, 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[Medline]
  5. Baum AE, Solberg LC, Kopp P, Ahmadiyeh N, Churchill G, Takahashi JS, Jameson JL, Redei EE 2005 Quantitative trait loci associated with elevated thyroid-stimulating hormone in the Wistar-Kyoto rat. Endocrinology 146:870–878[Abstract/Free Full Text]
  6. Dracopoli NC, Rettig WJ, Whitfield GK, Darlington GJ, Spengler BA, Biedler JL, Old LJ, Kourides IA 1986 Assignment of the gene for the ß subunit of thyroid-stimulating hormone to the short arm of human chromosome 1. Proc Natl Acad Sci USA 83:1822–1826[Abstract/Free Full Text]
  7. Szkudlinski MW, Teh NG, Grossmann M, Tropea JE, Weintraub BD 1996 Engineering human glycoprotein hormone superactive analogues. Nat Biotechnol 14:1257–1263[CrossRef][Medline]
  8. Grossmann M, Szkudlinski MW, Wong R, Dias JA, Ji TH, Weintraub BD 1997 Substitution of the seat-belt region of the thyroid-stimulating hormone (TSH) ß-subunit with the corresponding regions of choriogonadotropin or follitropin confers luteotropic but not follitropic activity to chimeric TSH. J Biol Chem 272:15532–15540[Abstract/Free Full Text]
  9. Matzuk MM, Kornmeier CM, Whitfield GK, Kourides IA, Boime I 1988 The glycoprotein {alpha}-subunit is critical for secretion and stability of the human thyrotropin ß-subunit. Mol Endocrinol 2:95–100[Abstract/Free Full Text]
  10. Grossmann M, Weintraub BD, Szkudlinski MW 1997 Novel insights into the molecular mechanisms of human thyrotropin action: structural, physiological, and therapeutic implications for the glycoprotein hormone family. Endocr Rev 18:476–501[Abstract/Free Full Text]
  11. Szkudlinski MW, Thotakura NR, Tropea JE, Grossmann M, Weintraub BD 1995 Asparagine-linked oligosaccharide structures determine clearance and organ distribution of pituitary and recombinant thyrotropin. Endocrinology 136:3325–3330[Abstract]
  12. Beck-Peccoz P, Persani L 1994 Variable biological activity of thyroid-stimulating hormone. Eur J Endocrinol 131:331–340[Abstract/Free Full Text]
  13. Yamada M, Saga Y, Shibusawa N, Hirato J, Murakami M, Iwasaki T, Hashimoto K, Satoh T, Wakabayashi K, Taketo MM, 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[Abstract/Free Full Text]
  14. Taylor T, Weintraub BD 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[Abstract/Free Full Text]
  15. Taylor T, Gesundheit N, Gyves PW, Jacobowitz DM, Weintraub BD 1988 Hypothalamic hypothyroidism caused by lesions in rat paraventricular nuclei alters the carbohydrate structure of secreted thyrotropin. Endocrinology 122:283–290[Abstract/Free Full Text]
  16. Biebermann H, Liesenkotter KP, Emeis M, Oblanden M, Gruters A 1999 Severe congenital hypothyroidism due to a homozygous mutation of the ßTSH gene. Pediatr Res 46:170–173[Medline]
  17. Doeker BM, Pfaffle RW, Pohlenz J, Andler W 1998 Congenital central hypothyroidism due to a homozygous mutation in the thyrotropin ß-subunit gene follows an autosomal recessive inheritance. J Clin Endocrinol Metab 83:1762–1765[Abstract/Free Full Text]
  18. Heinrichs C, Parma J, Scherberg NH, Delange F, Van Vliet G, Duprez L, Bourdoux P, Bergmann P, Vassart G, Refetoff S 2000 Congenital central isolated hypothyroidism caused by a homozygous mutation in the TSH-ß subunit gene. Thyroid 10:387–391[Medline]
  19. McDermott MT, Haugen BR, Black JN, Wood WM, Gordon DF, Ridgway EC 2002 Congenital isolated central hypothyroidism caused by a "hot spot" mutation in the thyrotropin-ß gene. Thyroid 12:1141–1146[CrossRef][Medline]
  20. Medeiros-Neto G, Herodotou DT, Rajan S, Kommareddi S, de Lacerda L, Sandrini R, Boguszewski MC, Hollenberg AN, Radovick S, Wondisford FE 1996 A circulating, biologically inactive thyrotropin caused by a mutation in the ß subunit gene. J Clin Invest 97:1250–1256[Medline]
  21. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M 2000 Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289:739–745[Abstract/Free Full Text]
  22. Morris AJ, Malbon CC 1999 Physiological regulation of G protein-linked signaling. Physiol Rev 79:1373–1430[Abstract/Free Full Text]
  23. Gether U 2000 Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr Rev 21:90–113[Abstract/Free Full Text]
  24. Vassart G, Dumont JE 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611[Abstract/Free Full Text]
  25. Vassart G, Pardo L, Costagliola S 2004 A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 29:119–126[CrossRef][Medline]
  26. Refetoff S 2003 Resistance to thyrotropin. J Endocrinol Invest 26:770–779[Medline]
  27. Refetoff S, Sunthornthepvarakul T, Gottschalk ME, Hayashi Y 1996 Resistance to thyrotropin and other abnormalities of the thyrotropin receptor. Recent Prog Horm Res 51:97–120; discussion 120–122[Medline]
  28. Moeller LC, Kimura S, Kusakabe T, Liao XH, Van Sande J, Refetoff 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[Abstract/Free Full Text]
  29. Govaerts C, Lefort A, Costagliola S, Wodak SJ, Ballesteros JA, Van Sande J, Pardo L, Vassart G 2001 A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor. J Biol Chem 276:22991–22999[Abstract/Free Full Text]
  30. Libert F, Lefort A, Gerard C, Parmentier M, Perret J, Ludgate M, Dumont JE, Vassart G 1989 Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem Biophys Res Commun 165:1250–1255[CrossRef][Medline]
  31. Parma J, Duprez L, Van Sande J, Hermans J, Rocmans P, Van Vliet G, Costagliola S, Rodien P, Dumont JE, Vassart G 1997 Diversity and prevalence of somatic mutations in the thyrotropin receptor and Gs {alpha} genes as a cause of toxic thyroid adenomas. J Clin Endocrinol Metab 82:2695–2701[Abstract/Free Full Text]
  32. Costagliola S, Bonomi M, Morgenthaler NG, Van Durme J, Panneels V, Refetoff S, Vassart G 2004 Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity. Mol Endocrinol 18:3020–3034[Abstract/Free Full Text]
  33. Van Sande J, Parma J, Tonacchera M, Swillens S, Dumont J, Vassart G 1995 Somatic and germline mutations of the TSH receptor gene in thyroid diseases. J Clin Endocrinol Metab 80:2577–2585[CrossRef][Medline]
  34. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2–{Delta}{Delta}C(T) method. Methods 25:402–408[CrossRef][Medline]
  35. Costagliola S, Khoo D, Vassart G 1998 Production of bioactive amino-terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor. FEBS Lett 436:427–433[CrossRef][Medline]
  36. Petersen G, Johnson P, Andersson L, Klinga-Levan K, Gómez-Fabre PM, Stahl F 2005 RatMap-rat genome tools and data. Nucleic Acids Res 33 (Database issue): D492–D494
  37. Weiss RE, Marcocci C, Bruno-Bossio G, Refetoff S 1993 Multiple genetic factors in the heterogeneity of thyroid hormone resistance. J Clin Endocrinol Metab 76:257–259[Abstract]
  38. Beck-Peccoz P, Persani L, Calebiro D, Bonomi M, Mannavola D, Campi I 2006 Syndromes of hormone resistance in the hypothalamic-pituitary-thyroid axis. Best Pract Res Clin Endocrinol Metab 20:529–546[CrossRef][Medline]
  39. Davies TF, Ando T, Lin RY, Tomer Y, Latif R 2005 Thyrotropin receptor-associated diseases: from adenomata to Graves disease. J Clin Invest 115:1972–1983[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. M. McLachlan, H. A. Aliesky, P. N. Pichurin, C.-R. Chen, R. W. Williams, and B. Rapoport
Shared and Unique Susceptibility Genes in a Mouse Model of Graves' Disease Determined in BXH and CXB Recombinant Inbred Mice
Endocrinology, April 1, 2008; 149(4): 2001 - 2009.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moeller, L. C.
Right arrow Articles by Weiss, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moeller, L. C.
Right arrow Articles by Weiss, R. E.
Right arrowPubmed/NCBI databases
*Gene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*LEVOTHYROXINE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals