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Departments of Medicine (L.C.M., X.-H.L., S.R.), Pediatrics (S.R.), and Committee on Genetics (S.R.), The University of Chicago, Chicago, Illinois 60637; Laboratory of Metabolism (S.K., T.K.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; and Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (J.V.S.), University of Brussels, School of Medicine, Erasmus Hospital, B 1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Samuel Refetoff, The University of Chicago, MC3090, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: refetoff{at}uchicago.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fourteen different mutations affecting the TTF1 gene have been reported in humans (3, 4, 5, 6, 7), but the clinical phenotype has been described in detail in only ten cases. Clear evidence for a clinical syndrome associated with TTF1 gene mutations was provided in 2002 with the report of six unrelated patients (5, 6). These mutations, which included missense, nonsense, or complete gene deletions, involved a single allele of the TTF1 gene. Yet, despite absence of dominant-negative effect of the mutant protein (5), the lack of one functional allele produced a clear phenotype. Neurological symptoms consisting of dyskinesia with or without hypotonia were present in all cases, and eight of 10 had history of respiratory distress. Although serum TSH concentrations were elevated in all, only five were clearly hypothyroid, showing serum T4 concentrations below the lower limit of normal. These findings in humans, suggesting TTF1 haploinsufficiency, prompted us to reexamine the heterozygous Ttf1 gene-deficient mice (Ttf1+/-). Indeed, the mice showed a similar phenotype of poor coordination and significantly elevated serum TSH concentration (5).
The mechanism responsible for the consistent elevation of TSH in the presence of a thyroid gland in a normal location in both human and mice remained unclear. The finding of pituitary and hypothalamic abnormalities in the homozygous mice, totally deficient in TTF1 (1), indicated the possibility of a central defect such as the production of a TSH molecule with reduced biological activity. Considering the importance of genes expressed in the thyroid gland and controlled by TTF1, TSH receptor (TSHr), thyroglobulin (TG), and thyroid peroxidase (TPO) (8), reduction of their expression due to TTF1 haploinsufficiency could also explain the observed hypothyroidism. We therefore decided to investigate the mechanism responsible for the thyroid abnormalities in Ttf1+/- mice by a systematic study of TTF1-controlled genes expressed in the thyroid gland, as well as the bioactivity of serum TSH and the thyroidal responsiveness to exogenous TSH. Our results confirm the presence of only half the amount of the TTF1 protein and show a reduced expression of the TSHr that is partially compensated by an increase in the serum TSH level. The mild hypothyroidism manifesting as reduced T4 and increase in TSH of normal bioactivity, could be overcome by administration of a larger amount of TSH
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RESULTS |
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). Mean serum TSH concentrations ± SE were 205 ± 22 and 92 ± 12 mU/liter in the Ttf1+/- and wild-type (WT, +/+) Ttf1+/+ mice, respectively (P < 0.001). The corresponding T4 concentrations were 47 ± 3 and 63 ± 6 nmol/liter (P < 0.02). Difference in absolute TSH value between this and our previous publication (5) is due to the inclusion of female mice that normally have reduced serum TSH levels (9).
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). The mean ratio of immunoreactive to bioreactive TSH (mU TSH/pmol cAMP) of 110 ± 10 and 120 ± 5 for Ttf1+/- and Ttf1+/+ mice was not different.
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, the T4 response to the low bTSH dose of 2mU was reduced in Ttf1+/- mice as compared with the Ttf1+/+ mice (a T4 increment of 5.5±2.2 vs. 15.3±4.1 nmol/liter; P < 0.04). However, administration of a larger dose of bTSH (10 mU) abolished this difference (17.4 ± 4.8 vs. 14.8 ± 7.8 nmol/liter; P > 0.5) (Fig. 2B). Together with the normal bioactivity of serum TSH, these results indicate that the defect responsible for the mild hypothyroxinemia in Ttf1+/- mice was at the level of the thyroid gland and that it could be overcome with increased amounts of TSH.
The thyroid glands of Ttf1+/- mice were of normal size, 2.2 ± 0.3 mg, as compared with 2.3 ± 0.4 mg in Ttf1+/+ mice and in agreement with our previous results (5). The absolute gland weight was slightly smaller due to the more careful dissection in order not to include any surrounding tissue. To confirm that TTF1 haploinsufficiency is responsible for the phenotype of Ttf1+/- mice, we quantified TTF1 at the mRNA and protein level. Because insertion of the 1.1-kb neo cassette in the homeodomain of the Ttf1 gene disrupts the production of normal TTF1 but not the formation of a transcript from the KO allele, we synthesized primers that amplify transcripts from both normal and disrupted alleles (total TTF1 mRNA) as well as primers specific for the intact TTF1 mRNA transcribed from the WT allele (see Table 1). As shown on Fig. 3, the amount of total transcript from both Ttf1 alleles was not different in theTtf1+/- mice from that in Ttf1+/+ mice. However, the amount of WT TTF1 mRNA in Ttf1+/- mice was half (54 ± 9%; P < 0.01) of that measured in Ttf1+/+ mice (Fig. 3A) or 48±4% the total transcript from both alleles (Fig. 3B). These and all subsequent mRNA values are expressed as percent of values in Ttf1+/+ animals being set to 100%. Western blot analysis confirmed this finding at the protein level (Fig. 4).
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). In contrast, differences between the two genotypes for PAX8, sodium iodide symporter (NIS) and type I iodothyronine 5'-deiodinase (DIO1) mRNAs were not significant (Fig. 5B).
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DISCUSSION |
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In our previous report, the slight reduction in mean level of serum T4 in Ttf1+/- mice did not reach statistical significance, even though the corresponding increase in serum TSH concentration was significant (P < 0.01). In the current group of animals, we not only confirm the presence of significant increase in serum TSH concentration (P < 0.001), but that the mild decrease in serum T4 level was also significant (P < 0.02). These results support the presence of a mild degree of hypothyroidism in the Ttf1+/- mice. Theoretically, this phenotype could be produced by either central hypothyroidism associated with reduction in the bioactivity of TSH (10, 11), or mild primary hypothyroidism caused by inherent defects in the thyroid gland. Because both are possible in animals with potential defects in the hypothalamus as well as in the thyroid gland, we tested both possibilities.
We showed that the increased TSH in serum of Ttf1+/- mice measured by RIA had normal bioactivity. In addition we determined the thyroidal responsiveness to biologically active TSH by measurement of T4 after the administration of a single dose of bovine TSH to Ttf1+/- and Ttf1+/+ animals. To eliminate the effect of endogenous TSH and to reduce the basal concentration of T4, animals were pretreated with L-T3. The almost 3-fold greater increase in serum T4 concentration in Ttf1+/+ than Ttf1+/- animals given the low (2 mU) dose of TSH indicates a reduced sensitivity of the thyroid gland to normal TSH, placing the defect at the level of the thyroid gland. This partial resistance to TSH was overcome by the administration of a larger dose (10 mU) of TSH.
Measurement of thyroidal mRNAs further delineated the pathophysiology of the thyroidal abnormality in Ttf1+/- mice. The amount of TSHr mRNA in Ttf1+/- mice was exactly half that in Ttf1+/+ mice. This reduction in TSHr is sufficient to explain the results observed after the administration of TSH. At low TSH level, the amount of saturated TSHr in Ttf1+/- animals is critically reduced and thus failed to produce a normal thyroidal response. However, a higher level of TSHr saturation, by an increased amount of TSH, fully compensated the defect as shown by the equal increment of T4 in Ttf1+/- and Ttf1+/+ animals (see Fig. 2B). Of the other two mRNAs, known to be controlled by TTF1 (8), only TG mRNA showed a statistically significant decrease in Ttf1+/- mice relative to Ttf1+/+ mice. This cannot be explained by the reduction in TSHr because recent work from Di Lauro’s laboratory (12), using TSH and TSHr deprived mice, showed that neither TSH nor its receptor had a significant effect on the amount of thyroidal TG.
TTF1 haploinsufficiency of Ttf1+/- mice did not affect the relative amount of PAX8, NIS, or DIO1 mRNA accumulation as compared with Ttf1+/+ mice. Although the effect of TTF1 on rodent PAX8 is not known, NIS is potentially regulated by TTF1 in the rat (13) and TTF1 has no effect on human DIO1 when tested in vitro, but species-specific differences have been observed (14). Although NIS expression in the mouse is dependent on the presence of TSHr, half the amount of TSHr found in heterozygous Tshr KO mice is sufficient to restore NIS production (15). This is compatible with our observations.
Thus, collectively, our results indicate that in the mouse, TTF1 haploinsufficiency produces hypothyroidism mainly through reduction in Tshr gene expression that is partially compensated by an increase in serum TSH. Whereas sufficient stimulation by TSH could fully correct the hypothyroidism, this could not occur in the untreated mouse, because maintenance of an increased endogenous concentration of TSH requires the stimulus provided by a reduced feedback suppression by thyroid hormone. Support for this interpretation can be obtained from recent data by Alberti et al. (16) showing that humans heterozygous for loss-of-function TSHr mutations have also increased serum TSH levels. The mean TSH ± SE levels for the eight-heterozygote individuals was 7.4 ± 1.1 mU/liter as compared with that of six normal relatives of 2.4 ± 0.3 mU (P < 0.002). This 3-fold increase in serum TSH in individuals expressing only one TSHr allele is remarkably similar to that of the Ttf1+/- mice also expressing half the TSHr. Their immunoreactive TSH was increased by 2.2-fold and bioreactive TSH by 2.7-fold as compared with the WT animals. Furthermore, as in the Ttf1+/- mice the increase in serum TSH could not fully correct the serum T4 levels. Although within the normal range, they are on the average 21% lower in humans heterozygous for loss-of-function TSHr mutations (mean of all published data) and by 25% lower in this study of Ttf1+/- mice. In contrast subjects heterozygous for TG gene defects have normal serum levels of TSH and T4 (17), in support to the minor effect of reduced TG levels on thyroid function.
How do our findings in mice relate to observations made in humans with TTF1 gene defects? Patients heterozygous for loss-of-function mutations in the TTF1 gene, present with variable degrees of hypothyroidism, ranging from subclinical to severe. As shown in Table 2, five of 10 patients were euthyroid, as judged by their thyroid hormone levels, three had mild to moderate hypothyroidism, and in two the hypothyroidism was severe. There is a good exponential reverse correlation between serum TSH and T4 concentrations (r = 0.857) and thyroid anatomy and degree of hypothyroidism. Indeed, euthyroid patients have normal thyroid glands, hypothyroid have hypoplastic glands, and the single patient with most severe hypothyroidism has apparent thyroid agenesis. In contrast, the extent of DNA defects does not correlate with the degree of thyroid abnormalities, nor with the severity of neurological or respiratory symptoms (Table 2; for details, see original publications). For example, patients with major chromosomal deletions, as well as those with nonsense mutations, showed the entire range of thyroid dysfunction. The one subject with a mutation producing a single amino acid substitution, had severe hypothyroidism with thyroid agenesis, as well as important neurological and respiratory symptoms. In euthyroid or mildly hypothyroid hyperthyrotropinemic patients, the haploinsufficiency of TTF1, resulting in a reduction in TSHr can explain the observed thyroid phenotype as that in the mouse; the elevated serum TSH level partially restoring thyroid function. However, there is no ready explanation, why in some patients with a similar gene defect, the hypothyroidism is more severe, and even higher serum TSH levels fail to improve thyroid function. Because these patients have thyroid hypoplasia, and even apparent aplasia, it is tempting to speculate that, as in the completely TTF1-deficient (-/-) mouse (1), these patients have, in addition, failure of glandular development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hormone Measurements
Serum TSH and T4 concentrations were measured by RIAs as previously described (9). TSH was also measured by bioassay using a line of Chinese hamster ovary cells stably transfected with a human TSH receptor cDNA, as previously described (21). The subclone cl 213 of JP26-26 used in this assay was particularly sensitive to low levels of TSH. Briefly, 50,000 cells were seeded in individual test tubes and incubated for 24 h in 100 µl Ham’s F-12 nutrient mixture supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate and 2.5 µg/ml Fungizone. Cells were washed with 500 µl Krebs Ringer HEPES buffer (pH 7.4), supplemented with 8 mM glucose and 0.5 g/liter BSA and then preincubated for 30 min in 200 µl of the same medium. The medium was removed and 200 µl of fresh buffer containing 20 µl of serum for TSH measurement and 25 µM Rolipram, a cAMP phosphodiesterase inhibitor, were added in duplicates. The incubation was continued for 1 h, at the termination of which the medium was discarded and replaced with 0.1 M HCl. cAMP was measured in the dried cell extract by RIA according to the method of Brooker et al. (22). Blanks were prepared as above but contained 20 µl of serum depleted of TSH by treatment of mice with 5 µg L-T4 for 10 d.
Thyroidal Response to TSH
A single injection of a low (2 mU) and a high (10 mU) dose bTSH (Sigma, St. Louis, MO) was given ip to animals pretreated for 4 d with 1 µg T3/d. Blood was obtained before and 3 h after bTSH injection for the measurement of T4. The optimal TSH dose and time of blood sampling were established by dose and time course experiments carried out in WT mice.
Tissues
Thyroid glands were dissected from 9 WT and 8 Ttf1+/- mice. RNA was extracted using phenol/guanidine isothiocyanate (TRIZOl Reagent, Life Technologies/Invitrogen, Carlsbad, CA) and was reverse transcribed with the SuperScript II ribonuclease (RNase) H Reverse Transcriptase (Life Technologies/Invitrogen) protocol using 2 µg of total RNA and 100 ng of random hexamers.
Real-Time PCR
Reactions for the quantification of TTF1, TPO, DIO1, PAX8, and TG mRNAs were performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA), using SYBR Green I as detector dye. 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. The sequences of all oligonucleotide primers are listed in Table 1.
The Ttf1 gene was knocked-out by insertion of a 1.1-kb neo-cassette into a XhoI site in the middle of the second exon, resulting in the disruption of the first helix of the homeodomain sequence (1). To measure specifically the expression of the WT and the disrupted allele we designed two primer pairs, one located in the 3'end to amplify both alleles (common, in Table 1) and another pair located just up- and downstream of the XhoI site used to insert the cassette to amplify only from the WT allele (WT-specific, in Table 1). Cycle conditions were 50 C for 2 min, 95 C for 15 min followed by 40 cycles of 95 C for 15 sec, 60 C for 30 sec and 72 C for 30 sec. The elongation time using the WT-specific primer pair was reduced to 10 sec.
Expression of TSHr mRNA was measured by the TaqMan assay principle, using a FAM/TAMRA-labeled probe. The reaction mixture contained 10 ng template cDNA, 12.5 µl TaqMan universal PCR master mix (Applied Biosystems/Roche, Branchburg, NJ), 0.25 µmol/liter of each primer, and probe and RNase-free water to a final volume of 25 µl. The sequences of the amplification and probe oligonucleotides are listed in Table 1. The reaction 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. Samples of untranscribed RNA used as template, showed no amplification with any primer pair, excluding amplification from contaminant genomic DNA. The expression of genes was calculated relative to that in the WT mice and normalized for 18S rRNA, measured under the same conditions with TaqMan Ribosomal RNA Control Reagent, VIC Probe (Applied Biosystems/Roche, Branchburg, NJ), using the 2-
CT method (23).
Western Blot Analysis
Nuclear and cytoplasmic extracts were prepared from pooled thyroid glands of WT and Ttf1+/- mice using nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL) according to the manufacturer’s instruction. Twenty micrograms of nuclear and cytoplasmic extracts were separated on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) using a Mini Trans-Blot Transfer Cell system (Bio-Rad, Hercules, CA). Membrane was preincubated for 1 h with 5% skim milk in PBS, followed by overnight incubation at 4 C with the primary antibody against TTF1 (Biopat, Caserta, Italy) at 1:1000 dilution with PBS containing 0.5% BSA. The membrane was washed in PBS, incubated for 1 h at room temperature with horseradish peroxidase-conjugated antirabbit IgG (Sigma) diluted 1:5000 and then washed with the same buffer. Protein bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposure to x-ray film. Protein concentration was determined with Bio-Rad protein assay reagents using BSA as standard.
Data Analysis
All results are expressed as mean ± SE and statistical analysis was done by ANOVA.
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FOOTNOTES |
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Abbreviations: bTSH, Bovine TSH; DIO1, type I iodothyronine 5'-deiodinase; KO, knockout; NIS, sodium iodide symporter; RNase, ribonuclease; TG, thyroglobulin; TPO, thyroid peroxidase; TSHr, TSH receptor; TTF1, thyroid transcription factor 1; WT, wild-type.
Received for publication May 12, 2003. Accepted for publication July 30, 2003.
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