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1 in Resistance to Thyroid Hormone: Study in Mice with a Targeted Mutation in the TRß Gene and Deficient in TR1
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264
Address all correspondence and requests for reprints to: Dr. Sheue-yann Cheng, Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Room 5128, Bethesda, Maryland 20892-4264. E-mail: sycheng{at}helix.nih.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 isoform in maintaining the normal functions of thyroid hormone (T3) in these patients. To understand the role of TR1 in the manifestation of RTH, we compared the phenotypes of mice with a targeted dominantly negative mutant TRß (TRßPV) with or without TR1. TRßPV mice faithfully recapitulate RTH in humans in that these mice demonstrate abnormalities in the pituitary-thyroid axis and impairment in growth. Here we show that the dysregulation of the pituitary-thyroid axis was worsened by the lack of TR1 in TRßPV mice, and severe impairment of postnatal growth was manifested in TRßPV mice deficient in TR1. Furthermore, abnormal expression patterns of T3-target genes in TRßPV mice were altered by the lack of TR1. These results demonstrate that the lack of TR1 exacerbates the manifestation of RTH in TRßPV mice. Therefore, TR1 could play a compensatory role in mediating the functions of T3 in heterozygous patients with RTH. This compensatory role may be especially crucial for postnatal growth. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and TRß, located on human chromosomes 17 and 3, respectively (1, 2). The four T3-binding TR isoforms, 1, ß1, ß2, and ß3, are derived from the TR and TRß genes 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 the thyroid hormone response elements (TREs) as a heterodimer or homodimer in the promoter regions of T3-target genes. The transcriptional activity of TR depends not only on the type of TRE but also on a host of corepressor and coactivator proteins (1, 2). Resistance to thyroid hormone (RTH) is a syndrome characterized by reduction in the sensitivity of tissues to the action of thyroid hormones and is caused by mutations of the TRß gene (4, 5). TRß mutants mediate the clinical phenotype by interfering with transcription of T3-regulated genes by means of a dominant-negative effect (6, 7). Most TRß mutants derived from RTH patients have reduced or a loss of T3-binding activity and transcriptional capacities (6, 7).
This disease is manifested by elevated levels of circulating thyroid hormones associated with insuppressible serum TSH. The other clinical features include short stature, decreased weight, tachycardia, hearing loss, attention-deficit hyperactivity disorder, decreased IQ, and dyslexia. RTH can appear in a sporadic form, but most commonly it is a familial syndrome with autosomal dominant inheritance. 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). A single patient homozygous for a mutant TRß has been reported (8), displaying an extraordinary and complex phenotype of extreme RTH with very high levels of thyroid hormones and TSH. Most patients are heterozygous with only one mutant TRß gene, and the clinical symptoms are mild (4, 5).
We have recently created a mouse model to understand the molecular basis of RTH. This was done by targeting the PV mutation to the TRß gene locus via homologous recombination (TRßPV mice) (9). The PV mutation was derived from a patient exhibiting severe RTH. It has a C-insertion in codon 448, which leads to a frame shift mutation in the last C-terminal 14 amino acids of TRß1. This change of the carboxyl-terminal sequence of TRß1 results in complete loss of T3-binding and TRE-mediated transcriptional activities (10). Similar to the clinical presentation observed in homozygous RTH patients (8), TRßPV/PV mice recapitulate severe dysfunction of the pituitary-thyroid axis, the result of which is an extraordinarily high TSH level despite highly elevated circulating thyroid hormones, impairment in weight gain, and delayed bone development (9). Consistent with heterozygous RTH patients, TRßPV/+ mice exhibit mild signs of resistance.
Most heterozygous RTH patients, however, are clinically euthyroid, a finding suggestive of a possible compensatory role of TR1 in maintaining the normal physiological functions of T3 in these patients. We crossed TRßPV mice with mice deficient in TR1 (11) and compared the phenotypes of TRßPV mice with or without TR1 to elucidate the role of TR1 in the manifestation of RTH. We found that the lack of TR1 worsened the dysregulation of the thyroid-pituitary axis in TRßPV mice and resulted in more severe impairment of postnatal growth. Furthermore, abnormal expression patterns of T3-target genes in TRßPV mice were altered by the lack of TR1. These results demonstrate that the lack of TR1 intensifies the manifestations of RTH in TRßPV mice. Thus, TR1 assumes a compensatory role in mediating the functions of T3 in heterozygous patients with RTH. This compensatory role may be essential for postnatal growth.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 in TRßPV Mice1 in the manifestation of RTH, we crossed dominantly negative TRß mutation (TRßPV) mice with mice carrying a TR1 null mutation (TR1-/-). As shown in Fig. 1A
, the mean serum total T4 (TT4) concentration in TRßPV/+TR1-/- mice (11.01 ± 0.32 µg/dl; n = 19) was 1.2-fold higher than that of TRßPV/+TR1+/+ mice (9.57 ± 0.35 µg/dl; n = 22; P < 0.01). The mean serum TT4 concentration in TRßPV/PVTR1-/- mice (68.53 ± 6.03 µg/dl; n = 7) was also 1.2-fold higher than that of TRßPV/PVTR1+/+ mice (59.53 ± 1.24 µg/dl; n = 16; P < 0.05); whereas the TR1-/- mice showed a 20% lower mean serum TT4 concentration (3.25 ± 0.22 µg/dl; n = 19) than did the wild-type mice (4.01 ± 0.26 µg/dl; n = 20).
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1 in TRßPV mice were observed in the serum total T3 (TT3) concentrations (Fig. 1B
). The mean serum TT3 concentration in TRßPV/+TR1-/- mice (3.52± 0.20 ng/ml; n = 22) was 1.3-fold higher than that of TRßPV/+TR1+/+ mice (2.69 ± 0.08 ng/ml; n = 22; P < 0.01). The mean serum TT3 concentration in TRßPV/PVTR1-/- mice (23.5 ± 1.98 ng/ml; n = 13) was 1.4-fold higher than that of TRßPV/PVTR1+/+ mice (17.2 ± 1.27 ng/ml; n = 19). No significant differences in TT3 were detected between TR1-/- (1.41 ± 0.06 ng/ml; n = 19) and wild-type mice (1.44 ± 0.07 ng/ml; n = 20).
The serum TSH level is a critical indicator of the severity of the dysfunction of the pituitary-thyroid axis. Consistent with our previous reports (9, 12), TRßPV/+TR1+/+ and TRßPV/PVTR1+/+ mice showed 1.7-fold and 535.3-fold higher mean serum TSH concentrations (31.29 ± 2.98 ng/ml; n = 18 and 9976.58 ± 935.53 ng/ml; n = 19, respectively) than did wild-type mice (18.64 ± 2.35 ng/ml; n = 17). As shown in Fig. 2A, the lack of TR1 in TRßPV mice further increased the serum TSH concentrations. The mean serum TSH concentration in TRßPV/+TR1-/- mice (44.47 ± 3.40 ng/ml; n = 14) was significantly higher (1.4-fold; P < 0.01) than that of TRßPV/+TR1+/+ mice. The mean serum TSH concentration in TRßPV/PVTR1-/- mice (16993.06 ± 2526.91 ng/ml; n = 11) was also 1.7-fold higher than that of TRßPV/PVTR1+/+ mice (P < 0.01).
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shows the ratios of the pituitary gland mass per gram body weight. Consistent with the extremely high serum TSH levels, the ratio for TRßPV/PVTR1+/+ mice was increased 1.9-fold as compared with that for wild-type mice (P < 0.01). Moreover, the ratio for TRßPV/PVTR1-/- mice was 2.2-fold higher than that for TRßPV/PV TR1+/+ mice (P < 0.0001). Thus, the pituitary weights correlated with serum TSH levels in that the serum TSH level of TRßPV/PVTR1-/- mice was increased 1.7-fold as compared with that of TRßPV/PV TR1+/+ mice (Fig. 2A). Taken together, these results indicate that the lack of TR1 in TRßPV/PV mice causes More severe resistance to the action of thyroid hormones. These results also indicate that the deficiency of TR1 worsens the dysregulation of the pituitary-thyroid axis in TRßPV mice.
Lack of TR1 Intensifies the Impairment of Postnatal Growth in TRßPV Mice
We have previously reported the impairment in postnatal growth in TRßPV/PV mice but not in TRßPV/+ mice (9, 12). To understand the possible effect of TR1 in the manifestation of growth impairment in TRßPV mice, we compared body weights of TRßPV mice with or without TR1 during a 12-wk postnatal period. Lack of TR1 virtually did not affect weight gain in the TR1-/- mice (Fig. 3A, left panel); however, the lack of TR1 significantly reduced the prepubescent growth spurt of TRßPV/+ mice beginning at 3 wk (1.13-fold; n = 23–25; P < 0.001) and continued until adulthood (12 wk; Fig. 3A, middle panel). More dramatic changes caused by the lack of TR1 were evident in TRßPV/PV mice in that 40% reduction in weight gain was apparent beginning at 3 wk of age and continuing to adulthood (n = 7–12; P < 0.001; Fig. 3A, right panel).
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1 did not affect weight gain in female TR1-/- mice (Fig. 3B, left panel). In the TRßPV/+ mice, however, the prepubescent growth spurt of TRßPV/+ mice was reduced by 3–5 wk of age due to the lack of TR1 (21%, 18%, and 8% reduction at 3, 4, and 5 wk of age, respectively, as compared with the wild-type siblings; n = 17–24; P < 0.001). A catch up in weight gain was observed as the wild-type female mice were reaching adulthood (6–12 wk; n = 17–24; Fig. 3B, middle panel). The reason for this sex-specific difference is not clear. This difference, however, was not detected in TRßPV/PV mice (Fig. 3B, right panel). The effect on the impairment of weight gain by the lack of TR1 was apparent beginning at 2 wk of age (21% reduction; n = 7–16; P < 0.05). At 3 wk of age, the reduction increased to 26% (P < 0.01), reached its peaked at 36% at 4 wk of age, and continued to adulthood (P < 0.001).
The deleterious effect of the lack of TR1 on growth was also apparent in the development of long bones, as shown by representative data in Fig. 4. The lengths of femora in TRßPV/+ and TRßPV/PV mice at 3.75 months of age were significantly shortened (5% and 17%, respectively; n = 6–8; P < 0.001). Taken together, our results indicate that the lack of TR1 manifested more severe impairment of postnatal growth in TRßPV mice, particularly in TRßPV/PV mice. These results suggest, at least in part, that the wild-type TR1 isoform plays a compensatory role in the postnatal development of TRßPV mice.
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1 prompted us to measure the serum IGF-I levels in adult mice (Fig. 5). Consistent with the severe growth impairment, the mean serum IGF-I concentration in TRßPV/PVTR1+/+ mice (519.57 ± 56.63 ng/ml; n = 8) was reduced 20% as compared with the wild-type mice (661.33 ± 39.16 ng/ml; n = 8; P < 0.0001). The lack of TR1 led to further reduction (58%) in the TRßPV/PVTR1-/- mice (218.82 ± 9.99 ng/ml; n = 8; P < 0.0001). The lack of TR1 did not affect the serum concentrations of IGF-I in the TR1-/- and TRßPV/+ mice (Fig. 5).
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11 on the abnormal gene regulation observed in TRßPV mice, we used quantitative real-time RT-PCR to compare the mRNA expression patterns of several T3-target genes in the tissues of TRßPV mice with or without TR1. Figure 6, A and B, shows the expressions of the TSH gene in the pituitary glands of wild-type, TR1-/-, and TRßPV mice with or without TR1. TSH consists of two polypeptides, the TSHß-subunit and the common -subunit (-SU), both of which are negatively regulated by T3. Consistent with our previous reports (12), the -SU mRNA in the pituitaries of TRßPV/PVTR1+/+ mice was abnormally up-regulated (8.8-fold) as compared with wild-type mice despite the highly elevated levels of thyroid hormones (Fig. 6A); this indicates the dominant-negative action of PV in the transcriptional regulation of -SU by the wild-type TR1. The increase in the abnormal expression of the -SU gene was caused by the lack of TR1 (1.6-fold more up-regulation in TRßPV/PVTR1-/- than in TRßPV/PVTR1+/+ mice: P < 0.0001). A slight reduction in the expression of the -SU gene in wild-type and TRßPV/+ mice was discerned by the lack of TR1, indicating an insignificant trend.
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shows that similar abnormal patterns were also observed in the expression of the TSHß gene; namely, an abnormal 64- and 81-fold up-regulation was observed in TRßPV/PVTR1+/+ and TRßPV/PVTR1-/- mice, respectively, as compared with wild-type mice. These data indicate that the lack of TR1 further increased the abnormal up-regulation of the TSHß gene 1.3-fold (P < 0.0001; Fig. 6B) in TRßPV/PV mice.
In addition, we also evaluated the effect of the lack of TR1 on the expression of GH gene in the pituitary. The expression of the GH gene is positively regulated by T3. However, instead of being activated by the elevated levels of T3 in TRßPV/PV mice, the expression of the GH gene was repressed (bar 5, Fig. 6C). Lack of TR1 led to 6.5-fold more repression of the GH in TRßPV/PVTR1-/- than in TRßPV/PVTR1+/+ mice (P < 0.05) and 1.1-fold more repression of GH in TRßPV/+TR1-/- than in TRßPV/+TR1+/+ mice (P < 0.01). These results indicate that lack of TR1 potentiates the in vivo dominant-negative effect of mutant TRß on the regulations of T3-target genes in the pituitary. The repression in the expression of GH is consistent with the reduced circulating IGF-I levels detected in TRßPV/PVTR1+/+ and TRßPV/PVTR1-/- mice (Fig. 5).
It has been shown that the predominantly expressed TR isoform in the heart is TR1 (11, 13, 14), which functions as the major TR to mediate the T3 action. We therefore determined the effect of the lack of TR1 in the expression patterns of T3-target genes in the heart, such as the hyperpolarization-activated cyclic nucleotide-gated channel (HCN) and the -myosin heavy chain (-MHC) that are the T3 positively regulated genes, and the ß-myosin heavy chain (ß-MHC) that is a T3 negatively regulated gene (14, 15). The lack of TR1 in the heart of TR1-/- and TRßPV/+ mice led to 40–50% repression in the expression of HCN2 mRNA (compare bars 2 and 4 with bars 1 and 3, Fig. 7A). The expression of HCN2 mRNA was increased 1.9-fold in TRßPV/PVTR1+/+ mice by the elevated circulating levels of thyroid hormone (bar 5, Fig. 7A), a finding that supports the premise that TR1 is the functioning TR mediating the activation of HCN2 mRNA. In the absence of TR1 in TRßPV/PVTR1-/- mice, the activated expression of HCN2 mRNA was abolished (compare bar 6 with 5, Fig. 7A). A similar pattern of response was also detected in the expression of the -MHC gene (Fig. 7B), but with a lower magnitude in the response. A 10% reduction was found in the TR1-/- mice (bar 2 vs. bar 1, Fig. 7B; P < 0.0001). A 1.3-fold activation in the expression of the -MHC gene was seen in the heart of TRßPV/PVTR1+/+ mice (bar 5 vs. bar 1, Fig. 7B) because of the elevated thyroid hormone. This activated expression was blocked when TR1 was absent in TRßPV/PVTR1-/- mice (bar 6 vs. bar 5, Fig. 7B). These data confirm that TR1 is the major TR that regulates the T3-target genes in the heart.
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1, the expression of the ß-MHC gene can no longer be repressed as evidenced by the abnormal up-regulation in TRß+/+TR1-/- mice (2.7-fold, P < 0.001; bar 2 vs. bar 1, Fig. 7C) and TRßPV/+TR1-/- mice (1.6-fold, P < 0.001; bar 4 vs. bar 3, Fig. 7C). Bar 5 shows that TR1 mediated the strong repression of the expression of the ß-MHC gene in TRßPV/PVTR1+/+ mice by the highly elevated thyroid hormone (60% repression as compared with the wild-type mice; bar 5 vs. bar 1, Fig. 7C). When TR1 was absent, the proper negative regulatory control was lost, resulting in an abnormal up-regulation of the ß-MHC gene (bar 6 vs. bar 5; 31.1-fold, Fig. 7C). Together, these findings indicate that TR1 functions to regulate the T3-target genes in the heart. In RTH, the abnormal regulation is due to the secondary effect of elevated thyroid hormones; however, the extent and patterns of regulation are target gene dependent. |
DISCUSSION |
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1 in the manifestation of RTH in vivo. A major role of thyroid hormone is to regulate its own production via the negative feedback loop acting on the hypothalamus-pituitary-thyroid (HPT) axis. The thyroid gland is stimulated by TSH, which is itself stimulated by hypothalamic TRH. Elevated levels of thyroid hormones suppress both TSH and TRH levels. Considerable progress has been made in the understanding of the role of TR isoforms in the regulation of the HPT by the use of mutant mice. The HPT axis in mice deficient in the TRß gene (TRß-/- mice) shows resistance to thyroid hormone, manifested as approximately 3-fold increased levels of TSH despite elevated T4 and T3 (16). Therefore, TRß is thought to play a major role in the negative feedback regulation of the HPT axis. The finding that administration of high doses of T3 to TRß-/- mice can partially suppress serum TSH level, but not below normal basal levels, indicates the necessity of TRß to achieve complete TSH suppression (17). In mice, a deficiency in both TR1 and TRß (TR1-/-TRß-/- mice) considerably exacerbates the TRß-/- phenotype (18), an indication that TR1 can also function in the negative feedback regulation of the HPT axis. Therefore, each TR pathway has not only specific roles but also redundant roles in the regulation of T3-target genes. The present study shows that the dysregulation of the pituitary-thyroid axis in both heterozygous and homozygous TRßPV mice (9) became more severe in mice deficient in TR1, suggesting a compensatory role of TR1 in the regulation of the pituitary-thyroid axis of RTH. These findings are entirely consistent with clinical observations in that many heterozygous RTH patients are euthyroid or display signs of mild dysregulation of the pituitary-thyroid axis (4, 5).
That no overt growth retardation was observed in mice lacking TR1 or TRß (16, 18) suggests the virtual overlap in the underlying TR1 and TRß pathways in the regulation of growth. The notion is further supported by the observations that adult mice deficient in both TR1 and TRß weighed approximately 30% less than the wild-type mice (18). Our data indicate that adult TRßPV/PV mice weighed 17–23% less than the wild-type mice (9). This impairment became more severe in TRßPV/PV mice lacking TR1 in that an additional approximately 40% reduction in body weight was detected in both the male and female mice (Fig. 3), another indication of the critical role of TR1 in the regulation of growth in RTH.
That TR1 functions to regulate growth in vivo is further supported by the reports that mice harboring mutations in the TR gene develop dwarfism (19, 20). Because the thyroid hormone can regulate growth indirectly by stimulating GH synthesis and because the growth-promoting action of GH is partly mediated by activation of the IGF-I synthesis, we also determined the serum IGF-I level and the GH mRNA expression. Similar to what was found in TR1-/-TRß-/- mice (18), both the serum IGF-I and GH mRNA were decreased in TRßPV/PV mice. In TRßPV/PV mice deficient in TR1, a further reduction in serum IGF-I and GH mRNA was observed in mice lacking TR1, indicating that TR1 plays a compensatory role in growth regulation via the common GH-IGF-I pathway.
The growth retardation observed in TRßPV mice was also reflected in the impaired development of the long bones. TR was found to be expressed in growth plate chondrocytes (21). Furthermore, we have recently demonstrated that TR1 is the major TR isoform in the long bones (femur and tibia) with a 10- to 12-fold higher expression than TRß1 (22). Thus, TRßPV mice deficient in TR1 exhibited more severe impairment (Fig. 4). Taken together, these findings emphasize that the postnatal growth in both TRßPV/+ and TRßPV/PV mice is, at least in part, compensated by TR1 via overlapping TR pathways. The critical T3-target genes contributing to postnatal growth remain to be identified.
Analysis of the abnormal regulation patterns of T3-target genes in TRßPV mice has helped uncover the importance of TR isoform distribution in tissue resistance in RTH. We have recently provided evidence to propose that differential expression of TR isoforms dictates the dominant-negative activity of mutant TRß and, therefore, the type of response in a given target tissue of RTH (13). In the present study, the effect of lack of TR1 on the response of T3-target genes is illustrated in two target issues with different TR isoform distribution. We examined the expression of the -SU and TSHß genes in the pituitary where TRß is the major TR isoform and the ß-MHC gene in the heart, where TR1 is the major isoform. These three genes are negatively regulated by T3. In the pituitary, instead of being repressed by the elevated thyroid hormones, the expression of -SU and TSHß genes was activated 8.8- and 64-fold, respectively, in TRßPV/PV mice. The lack of TR1 further increased the degree of abnormal up-regulation by 1.6-fold and 1.3-fold, respectively (Fig. 6). In the heart, however, a different response pattern emerges for the ß-MHC gene in TRßPV/PV mice. In mice with TR1, a normal repression response of the ß-MHC gene to the elevated thyroid hormone was detected. In TRßPV/PV mice deficient in TR1, however, a dramatic 31.1-fold activation was seen. These results highlight the importance of TR isoform distribution in the modulation of phenotypic expression by TR1.
In addition to TR isoform distribution, the contribution of the promoter context of T3-target genes in responding to PV and TR1 cannot be ignored. This additional effect is exemplified by the different sensitivity in the response of the two T3 positively regulated genes, the HCN2 and -MHC genes. The lack of TR1 led to a 50% reduction in the expression of the HCN2 gene in the presence of elevated thyroid hormone in the heart of TRßPV/+ mice, but virtually no reduction was discerned in the expression of the -MHC gene in the same mice. In the heart of TRßPV/PV mice, a 1.9-fold stimulation in the expression of the HCN2 gene in response to the elevated thyroid hormone was seen, whereas only a 1.3-fold increase in the activation of the -MHC gene was found. In TRßPV/PV mice deficient in TR1, the reduction in the activated expression was 80% and 50% for the HCN2 gene and the -MHC gene, respectively. Therefore, the HCN2 gene responds to the action of PV and TR1 with greater sensitivity. Elucidation of the underlying mechanisms for the differential sensitivity of these two genes awaits future studies.
In summary, our results show that the lack of TR1 significantly increased the resistance of target tissues to thyroid hormone in TRßPV mice. Therefore, TR1 plays an important compensatory role in maintaining the physiological functions of T3 in heterozygous patients with RTH. The present study illustrates that TRßPV mouse is a powerful tool not only to elucidate the molecular basis of RTH, but also help to clarify the in vivo functions of TR isoforms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1-deficient mutation, as previously described (9, 11). TRßPV mice (on a hybrid background of 129/Sv x C57BL/6 strains; Ref. 9) and TR1-/- mice (on a hybrid background of C57BL/6 and BALB/c strains; Ref. 11) were intercrossed several generations to generate wild-type, TR1-/-, TRßPV/+, TRßPV/+TR1-/-, TRßPV/PV, and TRßPV/PVTR1-/- mice for analysis. Phenotypic comparisons were made on siblings with the same mixed background.
Hormone Assays
The serum level of total T4 (TT4) and total T3 (TT3) were determined by using a Gamma Coat T4 and T3 assay RIA kit, (Dia-Sorin, Stillwater, MN), according to the manufacture’s instructions. Serum TSH levels were measured as previously described (9, 23). Serum IGF-I levels were determined by using a DSL-2900 Rat IGF-I RIA (Diagnostic Systems Laboratories, Webster, TX), according to the manufacturer’s instructions.
Quantitative Real-Time RT-PCR
Total RNAs were extracted from pituitaries and hearts of male mice at 3–4 months of age in each genotype using TRIzol (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Real-time RT-PCR for T3-target genes was performed employing a Roche Light Cycler PCR instrument and Light Cycler-RNA Amplification Kit SYBR Green I (Roche, Mannheim, Germany) with the specific primers as follows: GH, 5'-TTCGAGCGTGCCTACATT-3' (sense) and 5'-GCATGTTGGCGTCAAACTTG-3' (antisense); TSHß, 5'-GGATAGGAGAGAGTGTGCC-3' (sense) and 5'-AGCTTACGGCGACAGGGAA-3' (antisense); -MHC, 5'-CTGCGGAAACTGA-AAACGG–3' (sense) and 5'-TTCTTGCTACGGTCCCCTA-3'(antisense); ß-MHC, 5'-GACAGAGGAAGACAGGAAGA-3' (sense) and 5'-TGCTTTATTCTGCTTCCACCTA-3' (antisense); HCN2, 5'-ATGACCTACGACCTGGCAA-3' (sense) and 5'-AAGTCAGCGGGCAGTTTGT-3' (antisense); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ACATCATCCCTGCATCCACT-3' (sense) and 5'-GTCCTCAGTGTAGCCCAAG-3' (antisense). Total RNA (200 ng) was incubated at 55 C for 30 min and 95 C for 30 sec, followed by 45 PCR cycles, consisting of 95 C for 15 sec, 58 C for 30 sec, and 72 C for 30 sec. The crossing points were all below 30 cycles that are in the linear range of amplification.
Statistical Analysis
Data are expressed as mean ± SEM. Differences between groups were examined for statistical significance using Student’s t test or ANOVA with Fisher’s protected least significant difference post hoc test as appropriate.
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FOOTNOTES |
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-SU, -subunit; TRE, thyroid hormone response element; TRs, thyroid hormone receptors; TT3, total T3; TT4, total T4. Received for publication April 1, 2003. Accepted for publication May 5, 2003.
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REFERENCES |
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1. EMBO J 17:455–461[CrossRef][Medline]
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337 threonine thyroid hormone receptor ß mutant derived from the S family. Endocrinology 140:897–9021 receptor causes increased mortality, infertility and dwarfism in mice. Proc Natl Acad Sci USA 98:15095–151001. EMBO J 21:5079–5087[CrossRef][Medline]
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) was measured in male mice for each genotype at 3.75 months of age (n = 8–9). Each point represents a value for a single mouse and horizontal bars represent the mean values. **, P < 0.01; ***, P < 0.0001; N.S., not significant (P > 0.05).
