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Molecular Endocrinology, doi:10.1210/me.2002-0326
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Molecular Endocrinology 17 (5): 895-907
Copyright © 2003 by The Endocrine Society

Marked Potentiation of the Dominant Negative Action of a Mutant Thyroid Hormone Receptor ß in Mice by the Ablation of One Wild-Type ß Allele

H. Suzuki, X.-Y. Zhang, D. Forrest, M. C. Willingham and S.-Y. Cheng

Laboratory of Molecular Biology (H.S., X.-Y.Z., S.-Y.C.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264; Department of Human Genetics (D.F.), Mount Sinai School of Medicine, New York, New York 10029; and Department of Pathology (M.C.W.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Address all correspondence and requests for reprints to: Dr. S.-Y. Cheng, Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Room 5128, Bethesda, Maryland 20892-4264. E-mail: sycheng{at}helix.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the thyroid hormone receptor (TR) ß gene result in resistance to thyroid hormone (RTH), characterized by reduced sensitivity of tissues to thyroid hormone. To understand which physiological TR pathways are affected by mutant receptors, we crossed mice with a dominantly negative TRß mutation (TRßPV) with mice carrying a TRß null mutation (TRß-/-) to determine the consequences of the TRßPV mutation in the absence of wild-type TRß. TRßPV/- mice are distinct from TRß+/- mice that did not show abnormalities in thyroid function tests. TRßPV/- mice are also distinct from TRßPV/+ and TRß-/- mice in that the latter shows mild dysfunction in the pituitary-thyroid axis, whereas the former exhibit very severe abnormalities, including extensive papillary hyperplasia of the thyroid epithelium, indistinguishable from that observed in TRßPV/PV mice. Similar to TRßPV/PV mice, TRßPV/- mice exhibited impairment in weight gain. Moreover, the abnormal regulation patterns of T3-target genes in the tissues of TRßPV/- and TRßPV/PV mice were strikingly similar. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed with TR{alpha}1 for binding to thyroid hormone response elements in TRßPV/- mice as effectively as in TRßPV/PV mice. Thus, the actions of mutant TRß are markedly potentiated by the ablation of the second TRß allele, suggesting that interference with wild-type TR{alpha}1-mediated gene regulation by mutant TRß leads to severe RTH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THYROID HORMONE NUCLEAR receptors (TRs) are members of the steroid/retinoic acid receptor superfamily. They are thyroid hormone (T3)-dependent transcription factors that control growth, differentiation, development, and metabolic homeostasis (1, 2). Two TR genes, TR{alpha} and TRß, give rise to four T3-binding TR isoforms, ß1, ß2, ß3, and {alpha}1, by alternative splicing of the primary transcripts. Each TR isoform has unique developmental and tissue-specific patterns of expression (1, 3). TRs regulate transcription by binding to thyroid hormone response element (TRE) as a homodimer or heterodimer in the promoter region of T3-target genes. The transcriptional activity of TR depends on the types of TRE as well as the levels of T3. In the presence of T3, TRs function as transcriptional activators when bound to positive TREs, and as repressors when bound to negative TREs.

Resistance to thyroid hormone (RTH) is a syndrome characterized by the resistance of tissues to the action of thyroid hormones (4). RTH can appear in a sporadic form, but most commonly it is a familial syndrome with autosomal dominant inheritance. Most patients are heterozygotes with only one mutant TRß gene, and the clinical symptoms are mild (4, 5). Moreover, the clinical manifestations are variable between families with different mutations, between families harboring the same mutation, and also between members of the same family with identical mutations (4, 5). Some of the clinical features that have been reported include goiter, short stature, decreased weight, tachycardia, hearing loss, attention deficit-hyperactivity disorder, decreased IQ, and dyslexia (5, 6). One single patient homozygous for a mutant TRß has been reported (7). This patient displayed an extraordinary and complex phenotype of extreme RTH with very high levels of thyroid hormones and TSH. RTH is caused by mutations in the TRß gene (4, 5). TRß mutants derived from RTH patients have reduced or complete loss of T3-binding activity and transcriptional capacities. They act in a dominant negative fashion to cause the clinical phenotype (8, 9). Furthermore, RTH has been characterized in families in which TRß mutations have not been identified, suggesting that modifier genes could affect target organ responses to thyroid hormones (10).

To understand the molecular basis of RTH, we have recently created a mouse model by targeting the PV mutation to the TRß gene locus via homologous recombination (TRßPV mice; Ref. 11). The PV mutation was derived from a patient (P.V.) with severe RTH characterized by attention deficit-hyperactivity disorder, short stature, low weight, goiter, and tachycardia. PV has a unique mutation in exon 10, a C-insertion at the coding nucleotide position 1642 (codon 448), that results in the change of the last C-terminal 14-amino acid sequence of TRß1. This change of the sequence of TRß1 results in total loss of T3-binding and T3-dependent TRE-mediated transcriptional activities (12). Consistent with heterozygous RTH patients, heterozygous TRßPV/+ mice exhibit mild resistance in the pituitary-thyroid axis. Similar to the clinical presentation reported for the homozygous patient (7), homozygous TRßPV/PV mice manifest severe dysfunction of the pituitary-thyroid axis resulting in an extraordinarily high TSH level, despite highly elevated circulating thyroid hormones, growth impairment in weight gain, and delayed bone development (11).

Compared with other heterozygous family RTH patients in the same kindred (4, 5), the degree of severity and spectrum of resistance exhibited by the homozygous RTH patient could not simply be accounted for by doubling the mutant gene dose. We therefore hypothesized that the physiological consequences of the action of TRß mutant on TR isoforms (TRß and TR{alpha}1) differ. In the present study, we used TRßPV mice to test this hypothesis. We found that the phenotypes manifested by TRßPV/- mice and TRßPV/PV mice were strikingly similar, despite the differences in the number of mutant TRß alleles. In vivo, PV competed with TR{alpha}1 for binding to TREs in TRßPV/- mice as effectively as in TRßPV/PV mice. Furthermore, the abnormal regulation patterns of T3-target genes in TRßPV/- and TRßPV/PV mice were similar. Thus, the ablation of one wild-type TRß allele allows the mutant to more effectively compete with TR{alpha}1 for binding to TRE in the T3-target genes, thereby interfering with the transcriptional activity of TR{alpha}1, leading to a more severe phenotype. Thus, the present study provides direct evidence to indicate that TRß mutants can interfere with the transcriptional activity of TR{alpha}1 in vivo.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Severe Dysfunction of the Pituitary-Thyroid Axis in TRßPV/- Mice
We crossed TRßPV mice with mice deficient in two alleles of the TRß gene and performed thyroid function tests to assess the consequences of the ablation of the TRß gene allele on the action of the PV mutant. As shown in Fig. 1AGo, the lack of one wild-type TRß allele markedly enhanced the mean total T4 (TT4) concentration in TRßPV/- mice (34.48 ± 2.68 µg/dl; n = 30), that is 6.8-fold higher than that in the wild-type mice (5.18 ± 0.28 µg/dl; n = 19). In contrast, the lack of one wild-type TRß allele did not significantly affect TT4 concentrations in TRß+/- mice (4.97 ± 0.24 µg/dl; n = 19), and lack of two TRß alleles (8.07 ± 0.92 µg/dl; n = 19) only increased the TT4 to 1.5-fold higher than that of the wild-type mice. The TT4 level in TRßPV/- mice was significantly different from that in TRßPV/+ and TRß-/- mice (9.36 ± 0.45 µg/dl, n = 20; and 8.07 ± 0.92 µg/dl, n = 19, respectively, for TRßPV/+ and TRß-/- mice). An increase of 8.6-fold in TT4 was found in TRßPV/PV mice (44.38 ± 3.64 µg/dl; n = 16) as compared with the wild-type mice, which are slightly higher (~30%) than TRßPV/- mice.



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  		Figure 1. Comparison of Serum Thyroid Hormone Concentrations and Weights of Thyroids in TRßPV Mice with or without the Ablation of the Wild-Type TRß Gene

A, Serum levels of TT4; B, serum levels of TT3; C, weights of thyroid glands in adult mice (2–3 months old). Each point represents a value for a single mouse, and horizontal bars represent the mean values. *, P < 0.05; **, P < 0.01; ***, P < 0.001. N.S., Not significant. The fold change as compared with the wild-type mice is indicated.

 
Similar pattern changes caused by the lack of one wild-type TRß allele in TRßPV mice were observed in the serum total T3 (TT3) concentrations (Fig. 1BGo). The mean TT3 values for TRß+/-, TRß-/-, and TRßPV/+ mice were 1.05 ± 0.04 ng/ml (n = 20), 1.88 ± 0.12 ng/ml (n = 19), and 2.46 ± 0.07 ng/ml (n = 20), respectively. These TT3 concentrations represented small changes of 0.8-, 1.4-, and 1.8-fold increases for the TRß+/-, TRß-/-, and TRßPV/+, respectively, compared with that of the wild-type mice (1.37 ± 0.05 ng/ml; n = 19). In contrast, the lack of one wild-type TRß allele in TRßPV/- mice markedly enhanced the serum TT3 to 11.15 ± 0.8 ng/ml (n = 28), which is 8.1-fold higher than that in TRß+/+ mice (1.37 ± 0.05 ng/ml; n = 19). Consistent with our previous report (11), TT3 in TRßPV/PV mice (20.71 ± 3.3 ng/ml; n = 16) was 15.2-fold higher than that of the wild-type mice and 1.9-fold higher than that in TRßPV/- mice.

Consistent with the markedly elevated thyroid hormone levels, a 15.1-fold enlargement of the thyroid gland was observed in the TRßPV/- mice due to the lack of one wild-type TRß allele (Fig. 1CGo). In contrast, the lack of one wild-type allele reduced the weight of the thyroid gland by 20% in TRß+/- (2.1 ± 0.12 mg; n = 10) as compared with the wild-type mice (2.8 ± 0.18 mg; n = 10). The lack of two wild-type TRß alleles caused a small (1.6-fold; Fig. 2Go) increase in the weight of thyroid glands of TRß-/- mice (4.38 ± 0.49 mg; n = 10). Consistent with our previous report, the weight of thyroid glands in TRßPV/+ and TRßPV/PV mice was increased 1.6- and 20.8-fold, respectively (11). The extent of enlargement in the thyroid glands of TRßPV/PV mice was slightly larger (1.4-fold) than that in TRßPV/- mice.



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  		Figure 2. Comparison of Serum TSH Levels in TRßPV Mice with or without the Ablation of the Wild-Type TRß Gene

Serum TSH levels of adult mice (3–5 months old) were determined as described in Materials and Methods. Each point represents a value for a single mouse, and horizontal bars represent the mean values. **, P < 0.01; ***, P < 0.001. N.S., Not significant. The fold change as compared with the wild-type mice is indicated.

 
We also compared the histological features in the thyroids of wild-type and mutant mice at the same age (7 months). The thyroid glands of TRßPV/- mice revealed extensive papillary hyperplasia of the epithelium (data not shown) indistinguishable from that detected in the thyroid epithelium of TRßPV/PV mice reported previously (11). Taken together, except for minor quantitative differences in the levels of thyroid hormones, the results of thyroid function tests between TRßPV/- and TRßPV/PV mice were similar.

The circulating TSH levels also reflected the severity of the dysfunction of the pituitary-thyroid axis in TRßPV/- mice. Despite highly elevated levels of TT4 and TT3, TRßPV/- mice had 468.6-fold higher mean serum TSH concentration (8191.86 ± 440.77 ng/ml; n = 25) as compared with the wild-type mice (17.49 ± 2.10 ng/ml; n = 18). This markedly elevated TSH level was not significantly different from that of TRßPV/PV siblings, which showed a 554.2-fold higher serum TSH level (9687.27 ± 1300.70 ng/ml; n = 8). These increases were clearly distinct from those observed in TRßPV/+ and TRß-/- mice in which a mild 1.6-fold and 3.6-fold elevation of TSH was detected (27.48 ± 3.17 ng/ml, n = 20; and 63.56 ± 10.10 ng/ml, n = 11, respectively). Compared with TRß+/- mice in which the TSH concentration was not significantly different from that of the wild-type mice (17.20 ± 1.98 ng/ml; n = 20), the 3.6-fold increase of TSH in TRß-/- mice signifies an important regulatory role of the second TRß gene. These results indicate that similar to the TRßPV/PV mice (11), the pituitaries of TRßPV/- mice are extremely resistant to the action of elevated TT4 and TT3 (6.8- and 8.1-fold higher than that of the wild-type mice, respectively).

Using a mouse anti-TSH antibody, we also carried out immunohistochemical analyses of the pituitary glands to compare the histological changes in TSH-secreting cells in the wild-type and mutant mice. A significantly higher number of thyroprival cells, which are large cells stained to varying degrees with anti-TSH, were similarly observed in the pituitaries of TRßPV/- and TRßPV/PV mice (data not shown). Taken together, these data indicate that the severity of the resistance in the pituitary is indistinguishable between TRßPV/- and TRßPV/PV mice.

Whether there are gender differences in thyroid function tests was also assessed. As shown in Table 1GoGo, no gender differences in TT4 were observed in all genotypes except in wild-type mice in which a significant 30% higher concentration was found in male wild-type mice than in females. For TT3, gender differences were detected in wild-type, TRß+/-, and TRßPV/- mice in which the males had 1.2- to 1.5-fold higher values than the females. For TSH, gender differences were only observed in wild-type and TRß+/- mice in which males were found to have a 2-fold higher concentration than the females. Previously, higher TT4 and TSH levels in male mice than in female mice were also reported by Pohlenz et al. (13), and the reasons for such gender differences are currently unknown.


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  		Table 1. Gender Differences in Thyroid Function Tests

 

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  		Table 1A. Continued

 
Impairment in Weight Gain in TRßPV/- Mice
Previously, we found that TRßPV/PV mice exhibited impairment in body weight gain, in that TRßPV/PV mice are 17–23% smaller than their wild-type siblings at all ages examined, but no impairment in weight gain was detected in TRßPV/+ mice (11). The lack of one wild-type TRß gene together with one mutant allele led to the manifestation of growth impairment in female TRßPV/- mice (Fig. 3AGo). During the observation period of 4–12 postnatal weeks, TRßPV/- mice were significantly smaller (7–21%) than their wild-type littermates. The extent of impairment in weight gain shown in the growth curves was indistinguishable from that exhibited by TRßPV/PV mice (Fig. 3BGo). Thus, female TRßPV/- mice exhibit a similar growth defect similar to that observed for female TRßPV/PV mice (Fig. 3BGo).



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  		Figure 3. Impairment in Weight Gain of Female TRßPV/- Mice

The weight of wild-type, TRßPV/+, and TRßPV/- mice (A) and wild-type and TRßPV/PV mice (B) was measured in mice 4–12 wk of age. Data are shown as mean ± SE (n = 6–14). *, P < 0.01; **, P < 0.05, as compared with wild-type mice.

 
However, gender plays a role in the manifestation of this growth impairment. The lack of one wild-type TRß allele in TRßPV/- mice did not enhance the action of mutant PV in male TRßPV/- mice in that no growth impairment was detected (data not shown). The reasons for this gender difference are currently unknown.

Similarity in the Abnormal Expression Patterns of T3-Target Genes between TRßPV/- and TRßPV/PV Mice
The high degree of similarity in the phenotypes between TRßPV/- and TRßPV/PV mice suggested that T3-target genes could be similarly affected by the expression of mutant TRßPV in the tissues of TRßPV/- and TRßPV/PV mice. To find out whether this is the case, we compared the expression patterns of T3-target genes in the pituitary by Northern blot analyses. Figure 4Go shows the expression of TSH mRNA in the pituitary glands of wild-type and mutant mice. TSH consists of two polypeptides, the TSH-specific ß-subunit and the common {alpha}-subunit ({alpha}-SU), which are T3-negatively regulated genes. Instead of being repressed by the elevated levels of thyroid hormones, {alpha}-SU mRNA levels in the pituitaries of TRßPV/- and TRßPV/PV mice were abnormally 41.5- and 19.8-fold up-regulated as compared with the wild-type mice (Fig. 4Go, A and B). This abnormal regulation is not due to the ablation of the TRß gene (Fig. 4AGo, bar 2 vs. bar 1; bar 4 vs. bar 1) because virtually no change in the expression of {alpha}-SU mRNA was detected in TRß-/- and TRß+/- mice (Fig. 4Go, A and B). Similar patterns were also detected in the expression of the TSHß gene in that an abnormal 3.0- and 2.4-fold up-regulation was found in TRßPV/- (Fig. 4CGo, bar 5) and TRßPV/PV mice (Fig. 4CGo, bar 6; no significant differences between TRßPV/- and TRßPV/PV mice), respectively, and very little change was observed in TRß-/-, TRßPV/+, and TRß+/- mice (Fig. 4CGo, compare bars 2, 3, and 4 with bar 1). These results indicate that the extent of the in vivo dominant negative action of PV is similar between TRßPV/- and TRßPV/PV mice, despite the difference in the copy number of the mutant PV.



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  		Figure 4. Comparison of the Expression of the Common {alpha}-SU and TSHß Genes in the Pituitary of TRßPV Mice with or without Ablation of the Wild-Type TRß Gene

Northern blot analysis of {alpha}-SU mRNA (B) and TSHß mRNA (D) were determined using 3 µg/lane of total RNA from three adult male mice. Only the representative examples are shown. The loading of total RNA was normalized by stripping the blots and rehybridizing with [{alpha}-32P]-labeled GAPDH. After quantification, the relative mRNA expression is shown in panel A for {alpha}-SU and panel C for TSHß. Data are expressed as mean ± SE. *, P < 0.01; **, P < 0.0006. N.S., Not significant. The fold-change as compared with the wild-type mice is indicated.

 
The similarity in the expression patterns between TRßPV/- and TRßPV/PV mice was not limited to the T3-negatively regulated genes, but was also found in the T3-positively regulated genes. The GH gene is a T3-positively regulated gene in the pituitary. Instead of being activated by the elevated levels of thyroid hormones, the expression of the GH gene was abnormally repressed approximately 60% in TRßPV/- mice (data not shown), which was virtually identical to that observed previously in TRßPV/PV mice (11).

A similar abnormal regulation of another T3-positive regulated gene was found in type 1 deiodinase (D1) in the liver, in which the expression of D1 was too low to be detected in either TRßPV/- or TRßPV/PV mice despite 7- to 15-fold elevated thyroid hormones (data not shown). Taken together, these results indicate that the abnormal regulation patterns of T3-target genes are similar in TRßPV/- and TRßPV/PV mice, suggesting that the dominant negative action of one copy of the TRßPV gene in mice with the ablation of the other wild-type TRß allele is similar to that of two copies of the TRßPV gene.

TRßPV in the Nuclear Extracts of TRßPV/- Mice Competes with Wild-Type TRs for Binding to TRE in a Fashion Similar to TRßPV/PV Mice
As a first step to understanding the molecular basis of the similarity in the abnormal expression patterns of T3-target genes in TRßPV/- and TRßPV/PV mice, we prepared nuclear extracts from the livers of wild-type and mutant mice and compared their DNA binding patterns using EMSA. We chose to use Lys-TRE in EMSA because this TRE binds TRs with a high affinity so that the TR{alpha}1 expressed in low abundance in the liver can be detected for comparison (14, 15, 16). Lanes 1–6 of Fig. 5Go show the binding of Lys-TRE to the liver nuclear extracts of TRß+/+, TRßPV/+, TRßPV/PV, TRßPV/-, TRß+/-, and TRß-/- mice, respectively. Two major groups of bands (A and B) in each lane with different intensities and binding patterns were observed. Because of the close molecular sizes of TR isoforms and their heterodimeric partners (e.g. different RXR isoforms), it is impossible to clearly resolve and identify the nature of TRE-bound receptor species in these two major groups of bands.



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  		Figure 5. Binding of TR Isoforms and Mutant PV in the Liver Nuclear Extracts of Wild-Type and Mutant Mice to Lys-TRE

Equal amounts of nuclear extracts (12 µg) from different genotypes of mice as marked were incubated with [32P]-labeled TRE-Lys in the absence (lanes 1–6) or presence (lanes 7–24) of anti-TR isoform-specific antibodies or control antibody, MOPC (lanes 25–30), as described in Materials and Methods (IgG-ß1, 2 µg/lane; {alpha}1-403, 1:100 dilution/lane; MOPC, 2 µg/lane).

 
To circumvent this difficulty, we use TR isoform-specific antibodies (Fig. 5Go) to specifically super-shift TRE-bound TR isoforms. Consistent with our recent report (17), lanes 7–9 of Fig. 5Go show that wild-type TRß1 and PV were super-shifted by IgG-ß1, which specifically recognizes the amino terminus of both TRß1 and PV because they share the identical amino-terminus sequences. The gradual decrease in the intensities of the super-shifted bands in lanes 7, 8, and 9 suggests that the C-terminal region of PV, which has a totally different sequence from that of the TRß1, could weaken the interaction of the epitope in the A/B domain of PV with IgG-ß1 when compared with TRß1 (17). The band in lane 9 (Fig. 5Go) represented PV/IgG-ß1 complexes in TRßPV/PV mice without the contribution of TRß1 as in the TRßPV/+ mice (Fig. 5Go, lane 8). As expected, no super-shifted band was detected in TRß-/- mice (Fig. 5Go, lane 12), and furthermore the intensity of the super-shifted TRE-bound band in TRß+/- mice (Fig. 5Go, lane 11) was about half of that in TRß+/+ mice (Fig. 5Go, lane 7), confirming that the super-shifted bands in lanes 7–11 were Lys-bound TRß1 or PV bands. These results indicate that PV in TRßPV/- and TRßPV/PV mice bound to TRE.

Lanes 13–18 of Fig. 5Go show that TR{alpha}1 was specifically super-shifted by {alpha}1-403 with faster mobility than that of TRß1/IgG-ß1 or PV/IgG-ß1 complexes, which is consistent with the lower molecular weight of TR{alpha}1. It is known that the abundance of TR{alpha}1 protein is only about 20% that of TRß1 in the mouse liver (14, 17, 18). Therefore, only a small fraction of the TRE-bound TRs was super-shifted, and the majority of the TRE-bound TRs remained as faster mobility bands (bands A and B). These {alpha}1-403-super-shifted bands represented a mixture of {alpha}1-403 antibody complexed with TR{alpha}1/TR{alpha}1, PV/TR{alpha}1, or RXR/TR{alpha}1. It is, however, impossible to separate the individual contribution of each of these molecular species in these bands.

However, as shown in lanes 19–23, when both IgG-ß1 and {alpha}1-403 were used to super-shift TRE-bound TRs, most of the TRE-bound signals were now more retarded. In TRß-/- mice, only the Lys-TRE bound {alpha}1-403-super-shifted TR{alpha}1 signals were detected as expected (Fig. 5Go, lane 24). That the super-shifted bands detected in lanes 8–10 and 20–22 of Fig. 5Go contained TRE-bound mutant PV was further confirmed by using the anti-PV specific antibody T1 (19) in EMSA (data not shown). Lanes 25–30 of Fig. 5Go show that no super-shifted bands were detected when an irrelevant antibody, MOPC, was used, indicating the specificity of IgG-ß1 and {alpha}1-403 in super-shifting TRE-bound TRß1 and TR{alpha}1, respectively.

To assess whether PV competed with wild-type TRs as PV/TR{alpha}1 dimers in TRßPV/- mice in a similar fashion to TRßPV/PV mice, both T1 and {alpha}1-403 antibodies were used simultaneously to super-shift TRE-bound receptors in EMSA (Fig. 6Go). For easy comparison, lanes 1–6 show the TRE-bound super-shifted TR{alpha}1. Lane 7 of Fig. 6Go shows that an {alpha}1-403-super-shifted band with a similar intensity as that in lane 1 was detected in the wild-type mice. In TRßPV/+ mice, however, when both antibodies, T1 and {alpha}1-403, were present, a more retarded band (marked by {Delta}) in lane 8 of Fig. 6Go was detected. This more retarded band represented TR{alpha}1/PV heterodimers with {alpha}1-403 bound to the amino terminus of TRß1 and T1 bound to the C terminus of PV. In TRßPV/PV mice, the same super-shifted bands were detected (Fig. 6Go, marked by * and {Delta} in lane 9), but with approximately 2-fold increases in intensity (Fig. 6Go, lane 9 vs. lane 8). Importantly, the same two super-shifted bands with similar intensities as those in TRßPV/PV mice were detected in TRßPV/- mice (Fig. 6Go, lane 10 vs. lane 9). Consistent with the genotypes, only {alpha}1-403-super-shifted TRE-bound TR{alpha}1, but no T1 super-shifted bands, were detected in TRß+/- and TRß-/- mice (Fig. 6Go, lanes 11 and 12, respectively).



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  		Figure 6. Binding of TR Isoforms and Mutant PV in the Liver Nuclear Extracts of Wild-Type and Mutant Mice to Lys-TRE

Equal amounts of nuclear extracts (12 µg) from different genotypes of mice as marked were incubated with [32P]-labeled TRE-Lys in the presence (lanes 1–6) of anti-TR{alpha}1 antibody [{alpha}1-403 (1:100 dilution)] or in the presence of both T1 (lanes 7–12; T1, 2 µg/lane) and anti-TR{alpha}1 specific antibodies ({alpha}1-403, 1:100 dilution); or in the presence of anti-TR{alpha}1 specific antibodies ({alpha}1-403, 1:100 dilution) and anti-RXR antibodies (anti-RXR antibody, 1D-12: [1:100 dilution]/lane) as described in Materials and Methods.

 
To provide additional evidence to support the hypothesis that the more retarded bands in lanes 8, 9, and 10 (Fig. 6Go, marked by {Delta}) were TR{alpha}1/PV heterodimers, but not TR{alpha}1/RXR heterodimers, we performed super-shifted experiments in the presence of both anti-TR{alpha}1 antibody ({alpha}1-403) and RXR antibody (1D-12, recognizing all RXR isoforms; Fig. 6Go, lanes 13–18; Ref. 20). In TRß-/- mice with only TR{alpha}1, {alpha}1–403 antibody super-shifted TR{alpha}1 to the migration position as indicated by * in lane 12 (Fig. 6Go). When both anti-TR{alpha}1 antibody ({alpha}1-403) and anti-RXR antibody (1D-12) were used in the super-shifted experiments, a more retarded band (Fig. 6Go, marked by {circ} in lane 18) with a migration faster than that of the most retarded band shown in lanes 8, 9, and 10 (marked by {Delta}) appeared. This band was present in six genotypes that expressed TR{alpha}1 (Fig. 6Go, lanes 13–18). This band represented TRE-bound TR{alpha}1/RXR. The finding that TRE-bound TR{alpha}1/RXR migrated with a faster mobility than the more retarded bands in lanes 8, 9, and 10 further supports the conclusion that the more retarded band present in lanes 8, 9, and 10 (Fig. 6Go, marked by {Delta}) represented TR{alpha}1/PV heterodimers. Taken together, these results indicate that PV competes with TR{alpha}1 for binding to TRE in TRßPV/- mice as effectively as in TRßPV/PV mice (see Fig. 8Go, A and B).



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  		Figure 8. Schematic Representation of the Competition of Mutant PV with Wild-Type TR Isoforms for Binding to TRE

A1, In TRßPV/+ mice, PV competes with wild-type TRß and TR{alpha}1 for binding to TRE as PV/PV, PV/TRß, and PV/TR{alpha}1. A2, In TRßPV/- and TRßPV/PV mice, PV competes only with wild-type TR{alpha}1 for binding to TRE as PV/PV and PV/TR{alpha}1. B, Competition of mutant PV with wild-type TR isoforms for RXR in binding to TRE. B1, In TRßPV/+ mice, PV competes with wild-type TR isoforms for RXR (RXR/TRß and RXR/TR{alpha}1) in binding to TRE as PV/RXR. B2, In TRßPV/- and TRßPV/PV mice, PV competes only with wild-type TR{alpha}1 for RXR (RXR/TR{alpha}1) in binding to TRE as RXR/PV.

 
To understand whether PV in TRßPV/- mice also binds to TRE as a heterodimer with RXR, we used anti-RXR antibody (1D-12; Ref. 19). Lanes 7–12 of Fig. 7Go show that 1D-12 super-shifted TRE-bound receptors in the wild-type and the five mutant mice. Because RXRs do not bind to Lys-TRE (21), the 1D-12-super-shifted bands in lane 7 (Fig. 7Go; TRß+/+ mice) represented TR{alpha}1/RXR and TRß1/RXR dimers; in lane 8 (Fig. 7Go; TRßPV/+ mice), TR{alpha}1/RXR, TRß1/RXR, and PV/RXR; in lane 9 (Fig. 7Go; TRßPV/PV mice), TR{alpha}1/RXR and PV/RXR; in lane 10 (Fig. 7Go; TRßPV/- mice), TR{alpha}1/RXR and PV/RXR; in lane 11 (Fig. 7Go; TRß+/- mice), TR{alpha}1/RXR and TRß1/RXR; and in lane 12 (Fig. 7Go; TRß-/- mice), TR{alpha}1/RXR (see Fig. 8BGo). To confirm the formation of TRE-bound PV/RXR in TRßPV/+, TRßPV/PV, and TRßPV/- mice, we used both anti-PV (T1) and anti-RXR (1D-12) antibodies simultaneously in EMSA. As shown in lane 20 (Fig. 7Go; TRßPV/+ mice), a more retarded band (marked by {Delta}) than that in lane 14 super-shifted by T1 alone and that in lane 8 super-shifted by 1D-12 alone, was detected, indicating the formation of the PV/RXR dimers. As expected, no T1-super-shifted band was detected in lane 13 or in lane 19 from TRß+/+ mice. The formation of PV/RXR dimers was further confirmed by the increased intensity of PV/RXR dimer band in TRßPV/PV mice (Fig. 7Go, compare band marked by {Delta} in lane 21 with that in lane 15) and the concomitant reduction in the 1D-12 super-shifted band (Fig. 7Go, marked as *, compare with that in lane 9). These results indicate that PV also competes with TRs for RXR in binding to TRE (Fig. 8BGo).



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  		Figure 7. Competition of Binding of Mutant PV with TR Isoforms for Heterodimerization with RXR in Binding to Lys-TRE in the Liver Nuclear Extracts of Wild-Type and Mutant Mice

Equal amounts of nuclear extracts (12 µg) from different genotypes of mice as marked were incubated with [32P]-labeled TRE-Lys in the absence (lanes 1–6) or presence (lanes 7–24) of anti-PV specific antibodies and anti-RXR antibodies as marked as described in Materials and Methods (anti-RXR antibody, 1D-12: [1:100 dilution]/lane; T1, 2 µg/lane).

 
The formation of PV/RXR dimers was also apparent in TRßPV/- mice, as evidenced by the formation of a more retarded band (Fig. 7Go, marked by {Delta} in lane 22) super-shifted by both 1D-12 and T1. This was similar to that seen for TRßPV/PV mice (Fig. 7Go, the band marked by {Delta} in lane 21). This is further confirmed by the concomitant reduction in the intensity of the faster migrating band super-shifted by 1D-12 alone (Fig. 7Go, band marked by * in lane 22). Similar to the results seen in lanes 17 and 18, only wild-type TR/RXR complexes super-shifted by 1D-12 antibody were detected in lanes 23 and 24 (Fig. 7Go). Taken together, it is clear that PV also competes with w-TR{alpha}1 for RXR in binding to TRE in TRßPV/- mice (Fig. 8B2Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present study demonstrates the striking similarity in the severity of phenotypes manifested by two mutant mouse strains that retain two copies of TR{alpha}1, but that differed in the number of TRß mutant and wild-type alleles (TRßPV/PV and TRßPV/- mice). Except for minor quantitative differences in the serum concentrations of thyroid hormone, TRßPV/PV and TRßPV/- mice displayed similarly severe resistance in the pituitary thyroid axis with extraordinarily high TSH levels (468-fold increase). The intense proliferative thyroprival TSH cells were found to be diffusely distributed in the pituitaries of both mutant mice. Moreover, extensive papillary hyperplasia was similarly detected in the thyroids of TRßPV/PV and TRßPV/- mice. The impairment in weight gain was indistinguishable between TRßPV/PV and TRßPV/- mice. Consistent with an earlier report (22), we found that TRß-/- mice exhibit relatively mild resistance in the pituitary-thyroid axis, and no indication of resistance in the pituitary-thyroid axis was observed in TRß+/- mice. Therefore, the severe resistance is not simply caused by the ablation of the one wild-type TRß allele. These results indicate that the severity in RTH can be caused not only by the mutation of two PV alleles (TRßPV/PV mice) but also by mutation of one TRß mutant allele in the absence of one wild-type allele (TRßPV/- mice).

Having identified that an ablation of one wild-type TRß allele in TRßPV/- mice led to phenotypes similar to those of TRßPV/PV mice, we sought to understand the molecular basis of this similarity. We have shown earlier that in target tissues of TRßPV/PV mice, such as the liver and pituitary in which the major isoform is TRß, PV competes effectively with wild-type TR{alpha}1 for binding to TRE as TR{alpha}1/PV and PV/PV dimers (17). Furthermore, PV also competes with wild-type TR{alpha}1 for heterodimerization with RXR in binding to TRE (17). Based on the similarity in the phenotypes between TRßPV/PV and TRßPV/- mice, we expected that in TRßPV/- mice, PV would compete with TR{alpha}1 as effectively as in TRßPV/PV mice. Indeed, we found that in TRßPV/- mice, PV bound to TRE as strongly as that in TRßPV/PV mice (Fig. 6Go). The similar DNA binding patterns were also reflected in the abnormal regulation patterns of T3-target genes in tissues. D1, a T3-positively regulated gene in the liver, was similarly repressed in TRßPV/PV and TRßPV/- mice. The TSHß and GH genes in the pituitary were similarly activated and repressed, respectively, in TRßPV/PV and TRßPV/- mice, indicating similar potency of the dominant negative action of PV on the transcriptional activity of wild-type TR{alpha}1 between TRßPV/PV and TRßPV/- mice. Taken together, these findings indicate that one copy of the PV mutant is sufficient to cause as deleterious abnormalities as two copies of PV mutant do if the second TRß allele is absent.

Because only relatively mild resistance was observed in TRß-/- mice, it is clear that the ablation of the wild-type TRß allele in TRßPV/- mice is not directly responsible for the severe resistance observed. Rather, the absence of a wild-type TRß allele plays a permissive role to facilitate the interference of the mutant TRß with the transcriptional activity of TR{alpha}1. In the tissues of TRßPV/+ mice, in which TRß is the major isoform, PV competes with one copy of wild-type TRß and two copies of TR{alpha}1 for binding to TRE of the T3-target genes in tissues. Thus, the competition of PV with TR{alpha}1 for binding to TRE is less favorable in TRßPV/+ mice. In contrast, in TRßPV/- mice with no TRß allele, PV competes more effectively with TR{alpha}1 for binding to TRE as well as in TRßPV/PV mice that lack both wild-type TRß alleles (Figs. 6Go and 8Go). The similar phenotypes observed in TRßPV/PV and TRßPV/- mice suggest that effective competition of PV with TR{alpha}1 for binding to TRE leads to severe resistance by interference with the transcriptional activity of TR{alpha}1. It is important to point out that the mice lacking all known nuclear T3 receptors (TR{alpha}1-/- and TRß-/- mice; Ref. 23) exhibit many features that closely resemble the phenotypes of TRßPV/PV and TRßPV/- mice, indicating that the mice devoid of both TRß and TR{alpha} genes in the former and the interference of the functions of TR{alpha}1 by mutant PV in the latter results in severe phenotype. Thus, using TRßPV/- mice, the present study provides the direct evidence to indicate that mutant TRß can interfere with TR{alpha}1 functions in vivo.

Together with TRßPV/PV mice, the generation and characterization of TRßPV/- mice in the present study have helped uncover the important functional role of TR{alpha}1 in TRßPV/+ mice. The mild phenotypes in TRßPV/+ mice suggest that TR{alpha}1 could serve a redundant role for TRß when the actions of the wild-type TRß are preferentially affected by the dominant negative action of one mutated TRß. Alternatively, TR{alpha}1 plays a vital isoform-specific role to maintain most of the normal functions in TRßPV/+ mice. The redundant and isoform-specific roles of TR isoforms have been demonstrated using mice deficient in TRß, TR{alpha}1, or both TR{alpha}1 and TRß (23, 24). Thus, the severe phenotypes exhibited by TRßPV/PV and TRßPV/- mice could result from the interference with the redundant role and/or the vital isoform-specific actions of the TR{alpha}1 by the dominant negative action of PV. In this context, however, it is intriguing that the mutant TRß preferentially affects the functions mediated by wild-type TRß in TRßPV/+ mice. One possibility is via the preferential competition with wild-type TRß for binding to TRE by PV due to its higher abundance and higher affinity for the formation of inactive TRß/PV dimers (8, 9, 16). This notion is supported by the findings that PV heterodimerizes with TRß with a higher affinity than TR{alpha}1 (16). The other possibility is via isoform-dependent differential interaction with coregulatory proteins. It has been shown that lack of coactivator interaction can be a mechanism for dominant negative activity by mutant TRß (25, 26). It is entirely possible that the TRE-bound TRß/PV could be preferentially affected by the lack of coactivator interaction as compared with TR{alpha}1/PV in TRßPV/+ mice. In TRßPV/PV and TRßPV/- mice that both lack wild-type TRß, PV forms heterodimers with TR{alpha}1 (but no TRß to heterodimerize with PV), which can then be affected by the lack of coactivation interaction. It is important to point out that these two mechanisms are not mutually exclusive. The clarification will await future studies.

In summary, several independent assays of TR function have demonstrated that the action of mutant PV in inhibiting responses to thyroid hormone mediated by the TR is similarly effective in mice with two mutant alleles or with one mutant allele in the absence of the other wild-type allele. Mechanistically, we have demonstrated that PV in TRßPV/- mice competes effectively with TR{alpha}1 for binding to TREs leading to severe phenotypes. These studies emphasize the complexity of the regulation of gene expression involving a family of receptors and the utility of the detailed analysis of such dominant negative receptor isoforms. This novel mechanism for a dominant negative mutant observed in TRßPV/- mice could be applicable to other transcription factors or oncogenes for which isoforms are commonly coexpressed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mouse Strains
Mice were genotyped by PCR analysis using primers specific for the TRßPV mutation and the TRß-deficient mutation, as described (11, 22). TRßPV mice (on a hybrid background of 129/Sv x C57BL/6) and TRß-/- mice (a hybrid background of 129/Sv x C57BL/6J strains; Ref. 22) were intercrossed to generate wild-type, TRß+/-, TRß-/-, TRßPV/+, TRßPV/-, and TRßPV/PV on the same mixed background for analysis.

Preparation of Nuclear Extracts
Nuclear extracts were prepared by modification of the methods of Frain et al. (27) and Chamba et al. (28). Briefly, livers from mice were homogenized in buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.35 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, and Complete Mini EDTA-free (Roche Molecular Biochemicals, Mannheim, Germany)]. The homogenates were filtered through gauze and centrifuged at 700 x g for 10 min at 4 C. The nuclear pellets were resuspended in buffer A by gentle homogenization, layered over the same volume of buffer B (buffer A containing 0.5 M sucrose), and centrifuged at 1500 x g for 15 min. The washed nuclei were resuspended in buffer C (buffer A without sucrose), and centrifuged at 1500 x g for 10 min. The packed nuclei were resuspended in buffer D [20 mM HEPES (pH 7.9), 0.4 M KCl, 1.1 mM MgCl2, 5 mM DTT, 20% glycerol; Complete Mini EDTA-free and KCl were added to a final concentration of 0.55 M]. The nuclei were mixed gently for 30 min and centrifuged at 100,000 x g for 60 min at 4 C. The supernatant containing TRs was used for subsequent analyses as shown below.

EMSA
The double-stranded oligonucleotide containing the Lys-TRE was labeled with [{alpha}-32P]dCTP, similar to the method described by Zhu et al. (29). Nuclear extracts (12 µg) and approximately 0.2 ng of probe (3–5 - 104 cpm) was added to the binding buffer [25 mM HEPES (pH 8.0), 2 mM MgCl2, 0.01 mM ZnCl2, 5 mM DTT, 6% glycerol, 0.01% Triton X-100, nuclear extracts (12 µg), and 0.2 µg sheared salmon sperm DNA]. Binding reactions were performed at room temperature for 30 min in the presence or absence of appropriate antibodies with or without RXR. The complexes were resolved on 5.2% polyacrylamide gels in 0.5x TBE (45 mM Tris-HCl, 45 mM boric acid, 0.5 mM EDTA) at 250 V for 2.5 h. After drying of the gel, the DNA-bound proteins were detected by autoradiography.

Northern Blot Analyses
Total RNA was prepared from pituitary glands and livers with TRIzol (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. After electrophoresis, RNA was transferred onto membranes (Hybond-N+; Amersham Pharmacia Biotech, Boston, MA), which were hybridized with appropriate cDNA probes for glycoprotein {alpha}-SU, TSHß, GH, and D1. The blots were stripped and rehybridized with [{alpha}-32P]dCTP-labeled GAPDH cDNA. Quantification was performed using the Molecular Dynamics PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Hormone Assays
The serum levels of TT4 and TT3 were determined by using a Gamma Coat T4 and T3 assay RIA kit, respectively (Dia-Sorin, Inc., Stillwater, MN), according to the manufacturer’s instructions. TSH levels in serum were measured as described (11, 13, 30).

Histology and Immunostaining
Thyroid glands were fixed in 10% neutral buffered formalin, fixed, and subsequently embedded in paraffin. Five-micrometer-thick sections were prepared for staining with hematoxylin and eosin. Pituitaries were fixed in 10% formalin in PBS for 2 h at 23 C, dehydrated, and embedded in paraffin. Five-micrometer-thick sections were prepared for immunostaining with antimouse TSH antibodies (Biogenesis, Inc., Kingston, NH; Ref. 11).

Data Analysis
All data are expressed as mean ± SE. Statistical analysis used the Student’s t test, and a P value less than 0.05 was considered significant unless otherwise indicated.


   FOOTNOTES
 
D.F. is supported by NIH Grant DC-03441, the March of Dimes Birth Defects Foundation, and the Hirschl Trust.

Abbreviations: 1D-12, Anti-RXR antibody; D1, type 1 deiodinase; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RTH, resistance to thyroid hormone; {alpha}-SU, {alpha}-subunit; T1, anti-PV antibody; TR, thyroid hormone receptor; TRßPV mice, targeted PV mutation to the TRß gene locus; TRE, thyroid hormone response element; TT3, serum total T3; TT4, serum total T4.

Received for publication September 17, 2002. Accepted for publication January 29, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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