Differential Expression of Thyroid Hormone Receptor Isoforms Dictates the Dominant Negative Activity of Mutant ß Receptor

Xiao-Yong Zhang, Masahiro Kaneshige, Yuji Kamiya, Kumiko Kaneshige, Peter McPhie and Sheue-Yann Cheng

Gene Regulation Section (X.-Y.Z., M.K., Y.K., K.K., S.-Y.C.), Laboratory of Molecular Biology, National Cancer Institute, and Laboratory of Biochemical Pharmacology (P.M.), National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Sheue-yann Cheng, Laboratory of Molecular Biology, Building 37, Room 5128A, National Cancer Institute, 37 Convent Drive, MSC 4264, 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 ß gene (TRß) cause resistance to thyroid hormone (RTH). Genetic analyses indicate that phenotypic manifestation of RTH is due to the dominant negative action of mutant TRß. However, the molecular mechanisms underlying the dominant negative action of mutants and how the same mutation results in marked variability of resistance in different tissues in vivo are not clear. Here we used a knock-in mouse (TRßPV mouse) that faithfully reproduces human RTH to address these questions. We demonstrated directly that TRß1 protein was approximately 3-fold higher than TR{alpha}1 in the liver of TRß+/+ mice but was not detectable in the heart of wild-type and TRßPV mice. The abundance of PV in the liver of TRßPV/PV was more than TRßPV/+ mice but not detectable in the heart. TR{alpha}1 in the liver was approximately 6-fold higher than that in the heart of wild-type and TRßPV mice. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed not only with TR isoforms for binding to thyroid hormone response elements (TRE) but also competed with TR for the retinoid X receptors in binding to TRE. These competitions led to the inhibition of the thyroid hormone (T3)-positive regulated genes in the liver. In the heart, however, PV was significantly lower and thus could not effectively compete with TR{alpha}1 for binding to TRE, resulting in activation of the T3-target genes by higher levels of circulating thyroid hormones. These results indicate that in vivo, differential expression of TR isoforms in tissues dictates the dominant negative activity of mutant ß receptor, thereby resulting in variable phenotypic expression in RTH.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THYROID HORMONE RECEPTORS (TRs) are ligand-dependent transcription factors that mediate the biological activities of thyroid hormone (T3) (1). Four T3 binding TR isoforms have been identified, TR{alpha}1, TRß1, TRß2, and TRß3 (1, 2), which are derived from the TR{alpha} and TRß genes, respectively. Each TR isoform has a unique developmental and tissue-specific expression. TR binds to specific DNA sequences known as thyroid hormone response elements (TRE) in the promoter regions of T3 target genes. The transcriptional activity of TR is regulated by T3, by the type of TRE and by a host of coregulatory proteins (1, 3).

Resistance to thyroid hormone (RTH) is a syndrome caused by mutations of the TRß gene (4) and characterized by elevated levels of circulating thyroid hormone associated with normal or high levels of serum TSH (thyrotropin) (4). RTH can appear in a sporadic form, but most commonly it is a familial syndrome with autosomal dominant inheritance (4). Most patients are heterozygotes with only one mutant TRß gene. However, one single patient homozygous for a mutant TRß has been reported (5). Most TRß mutants derived from RTH patients have reduced T3-binding affinities and transcriptional capacities. Genetic analyses indicate that TRß mutants act in a dominant negative fashion to cause the clinical phenotype (4, 6).

Using cultured cells and reporter systems, several transfected TRß mutants were shown to repress TRE-reporter activity, providing in vitro evidence to support the dominant negative action of TRß mutants (7, 8). Based on these studies, three possible mechanisms were proposed to account for the dominant negative activity: 1) formation of inactive dimers between mutant and wild-type TRs (w-TRs); 2) competitions of mutant with w-TRs for binding to TRE; and 3) competition for limiting amounts of auxiliary proteins, such as the retinoid X receptors (RXRs) (7). Using in vitro translated w-TRs and several TRß mutants including PV, formation of TRE-bound mutant/TRß1, mutant/TR{alpha}1, mutant/mutant and mutant/RXR were demonstrated by EMSA (7, 8). These in vitro studies suggest that all three mechanisms could mediate the dominant negative action of mutant TRß in vivo.

However, how TRß mutants act to exert the dominant negative action in vivo is still unclear. The availability of a newly developed knock-in mouse model (TRßPV mouse) provides us with a valuable tool to examine the competition model in vivo. Furthermore, using this mouse, we also sought to understand how the differential expression of TR isoforms in tissues affects the competition of mutant TR with w-TRs leading to different expression patterns of T3-target genes, thereby resulting in different clinical phenotypes. TRßPV mouse was created by introducing the PV mutation into the TRß gene locus by homologous recombination (9). PV mutant was derived from an RTH patient with a C-insertion in codon 448, which leads to a frame shift mutation in the carboxy-terminal 16 amino acids of TRß (10). TRßPV knock-in mice faithfully reproduce human RTH in that they exhibit the hallmarks of elevated levels of circulating thyroid hormones and thyrotropin, goiter, and growth abnormality (4, 9).

In the present study, we developed a method to determine directly the protein abundance of TRß1 and TR{alpha}1 in the liver and the heart. We found that TRß1 was expressed approximately 3-fold higher than TR{alpha}1 in the liver, and it was more than 10-fold higher than that in the heart. TR{alpha}1 was expressed approximately 6-fold higher in the liver than in the heart. These data are further validated by the functional T3 binding assays. Using TR isoform- and PV-specific antibodies in EMSA, we demonstrated that in vivo, PV competed with w-TR for binding to TRE and for heterodimerization with RXRs. The extent of competition was dictated by differential expression of these two TR isoforms in the tissues. Thus, in the liver where the expression of TRß protein was more abundant than TR{alpha}1, the T3-positively regulated genes malic enzyme (ME), spot 14 (S14), and deiodinase 1 (D1) were repressed due to an effective competition by the abundantly expressed PV. In the heart, the significantly lower PV was unable to compete with w-TR{alpha}1 for binding to T3-target genes; therefore, appropriate expression of T3 target genes was manifested in the presence of higher level of circulating thyroid hormones.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Expression of TRß1 and TR{alpha}1 Proteins Is Tissue Dependent
Previously it has been shown that the ratios of expression of TR isoforms at the mRNA level varies from tissue to tissue (1, 11). Less is known about their expression ratios at the protein level due to the difficulty in detecting the low amounts of receptor proteins in tissues. Using isoform-specific antibodies and subtractive T3 binding activities, Schwartz et al. (11) estimated the expression of TR isoform proteins in rat tissues. Except for the liver (12), whether the same distribution patterns also occur in mouse tissues is unclear.

To determine the TR proteins in mouse tissues, we have now devised a two-step procedure to circumvent the difficulty in the direct detection of TR proteins due to low abundance. Nuclear extracts of the liver of wild-type or TRßPV mice were first immunoprecipitated with antibodies specific for the w-TRß1 or mutant PV followed by Western blot analysis. For the immunoprecipitation of w-TRß1, antibody IgG-ß1 whose epitope is located in amino acids 62–92 of the A/B domain of TRß1 was used (Fig. 1Go, A and B) (11). For Western blot analysis, we used monoclonal antibody C4 (mAb C4) whose epitope is located in the C-terminal 457EVFED461 of TRß1 (Fig. 1Go, A and B) (13). We found a higher level of TRß1 (~2-fold) in the liver of TRß+/+ mice compared with TRßPV/+ mice (lanes 3 vs. 2, Fig. 2AGo). Both the full-length (band a) and the truncated TRß1 (band b) were detected (lanes 2 and 3, respectively) (11, 14). No TRß1 was detected in the liver of TRßPV/PV mice (lane 4, Fig. 2AGo) indicating that, consistent with the genotype (9), no TRß1 was expressed. Lane 1 of Fig. 2AGo shows the control in which TRß1 prepared from in vitro transcription/translation was treated identically. Lanes 5–7 show that no protein bands were detected when an equal amount of normal rabbit IgG was used as a control for immunoprecipitation, indicating that the TRß1 bands detected in lanes 2–3 of Fig. 2AGo were specific. The control for protein loading was demonstrated by using Western blot analysis of the nuclear extracts using antiactin antibody (Fig. 2BGo). Figure 2BGo shows that the loading of the proteins was similar as indicated by the equal amounts of the actin detected.



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Figure 1. The Sequences (A) and Locations (B) of the Epitopes for the Anti-TR{alpha}1, Anti-TRß1, or Anti-PV Antibodies

 


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Figure 2. Expression of TRß1 and PV Proteins in the Liver of TRßPV Mice

A, Liver nuclear extracts (1 mg) of TRßPV mice or 10 µl of in vitro translated TRß1 (lane 1, control, "c") were immunoprecipitated using 10 µg of rabbit polyclonal anti-TRß1 (IgG-ß1) (lanes 1–4) or normal rabbit IgG (lanes 5–7) according to Materials and Methods. The immunoprecipitated proteins were analyzed by Western blotting using monoclonal anti-TRß1 antibody, C4, (2 µg/ml) as described in Materials and Methods. B, Nuclear extracts (20 µg) were loaded onto a 10% SDS polyacrylamide gel. After being transferred to PVDF membrane, actin was detected using antiactin antibody (0.2 µg/ml) as described in Materials and Methods. C, Liver nuclear extracts (2 mg) of TRßPV mice were immunoprecipitated using 10 µg of monoclonal anti-PV antibodies, mAb 302 (lanes 1–3) or 10 µg of an irrelevant monoclonal antibody, MOPC (lanes 4–6). The immunoprecipitated proteins were analyzed by Western blotting using T1 (0.5 µg/ml) similarly as described above. The genotypes of the mice are marked.

 
To determine the abundance of PV protein in the liver of TRßPV mice, we used the recently developed monoclonal anti-PV antibody, mAb 302, whose epitope is located in the C-terminal 16 amino acids of the PV protein (aa 447–463) (Fig. 1Go, A and B) to first immunoprecipitate PV protein, but not TRß1, followed by Western blot analysis using polyclonal anti-PV antibody, T1 (Fig. 1Go, A and B) (13). The sequences of the C-terminal 16 amino acids of TRß1 and PV are different due to a frame-shift mutation in the TRß gene (10). Using these antibodies in the two-step procedure, we expected to detect only PV protein in the liver. Indeed, lanes 2 and 3 of Fig. 2CGo show a specific band with a molecular mass of 55 kDa (band a) was detected in the liver of TRßPV/+ and TRßPV/PV mice, but not in TRß+/+ mice (lane 1 of Fig. 2CGo). Similar to TRß1 shown in lanes 2 and 3 of Fig. 2AGo, a truncated PV was also detected (band b in lanes 2 and 3 of Fig. 2CGo). Importantly, the intensities of intact and truncated PV in TRßPV/PV mice (lane 3 of Fig. 2CGo) was approximately 2-fold higher than that in TRßPV/+ mice (lane 2, Fig. 2CGo). When the nuclear extracts of wild-type and TRßPV mice were first immunoprecipitated with an irrelevant monoclonal antibody against an unknown antigen, MOPC, only very weak background bands were detected (lanes 4–6, Fig. 2CGo), indicating that the PV protein bands detected in lanes 2 and 3 of Fig. 2CGo were specific. Taken together, these results indicate that in TRßPV/PV mice, PV protein but not TRß1 was expressed, which is consistent with the genotype.

We carried out a similar analysis in the hearts of TRß+/+, TRßPV/+, and TRßPV/PV mice to evaluate the expression of TRß1 and PV proteins. However, using identical amounts of nuclear extracts (1 mg) as in the liver, no TRß1 protein was detected in the heart (data not shown). A 10-fold increase in the nuclear extracts still was not adequate to yield enough TRß1 protein to be detected under the same experimental conditions (lane 3 vs. lane 2, Fig. 3AGo). Increasing nuclear extracts yielded only an increase in the nonspecific proteins as evident in lane 4 (Fig. 3AGo) in which normal rabbit IgG was used for the immunoprecipitation. The identical amounts of actin detected in lanes 3 and 4 of Fig. 3BGo indicate that the loading of proteins in lanes 3 and 4 of Fig. 3AGo was identical. These results indicate that the expression of TRß1 protein in the heart was at least less than 10% of that in the liver. The abundance of PV proteins was also too low to be detected in the heart of TRßPV/PV mice (data not shown).



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Figure 3. TRß1 Protein Was Not Detectable in the Heart of TRß+/+ Mice

A, Liver nuclear extract (1 mg; lane 2), heart nuclear extract (10 mg; lanes 3–4) or 10 µl of in vitro translated TRß1 (lane 1) were immunoprecipitated using 10 µg of IgG-ß1 (lanes 1–3) or normal rabbit IgG (lane 4). The immunoprecipitated proteins were analyzed by Western blotting using mAb C4 (2 µg/ml) as described in Materials and Methods. B, Nuclear extracts of the liver (10 µg; lane 2) and of the heart (100 µg; lanes 3 and 4) were loaded onto a 10% SDS polyacrylamide gel. After being transferred to PVDF membrane, actin was detected using antiactin antibody (0.2 µg/ml) as described in Materials and Methods.

 
We further compared the relative expression levels of TR{alpha}1 and TRß1 proteins in the liver of wild-type and TRßPV mice. For immunoprecipitation, we used mAb C4 that recognizes both TRß1 and TR{alpha}1 proteins due to the presence of the identical C-terminal epitope, EVFED (Fig. 1Go, A and B) (13). For Western blot analysis, we used polyclonal antibodies that specifically recognize TR{alpha}1 protein ({alpha}1–403; FigGo. 1, A and B) (Fig. 4A-aGo) (15), TRß1 protein (IgG-ß1) (Fig. 4BGo) or both TR{alpha}1 and TRß1 proteins (C91) (Fig. 4CGo). C91 is polyclonal rabbit antipeptide antibody that recognizes both TR{alpha}1 and TRß1 proteins (Fig. 1Go, A and B) (14). Lane 1 of Fig. 4A-aGo shows the molecular marker for [35S]-labeled TR{alpha}1 that was prepared by in vitro translation. A full-length (band a) and truncated TR{alpha}1 (band b; derived from a downstream ATG) with apparent molecular masses of 47 and 43 kDa, respectively, were observed. Repeated experiments indicate that the ratios of bands a and b remained constant under identical experimental conditions. Lane 2 of Fig. 4A-aGo shows the molecular marker for [35S]-labeled TRß1 that was prepared by in vitro translation. In addition to the full-length-TRß1, a truncated TRß1 derived from a downstream ATG with an apparent molecular mass of 52 kDa was observed (14). Lane 3 shows that full-length and truncated TR{alpha}1 were specifically detected by anti-TR{alpha}1 antibodies after nuclear extracts of the liver of TRß+/+ mice (2 mg) were first immunoprecipitated by mAb C4. Lane 4 shows that more TR{alpha}1 was detected when an increased amount of nuclear extracts (5 mg) were used. When the same amounts of nuclear extracts (2 and 5 mg for lanes 5 and 6, respectively) were similarly immunoprecipitated by an irrelevant antibody, MOPC, only a weak nonspecific band was detected. Lane 7 shows that when a similar amount of nuclear extract from the heart of TRß+/+ mice as that in the liver (lane 4; 5 mg) was used, no TR{alpha}1 protein was detectable, indicating that the abundance of TR{alpha}1 protein was significantly lower in the heart. The control for protein loading was demonstrated by using Western blot analysis of the nuclear extracts using antiactin antibody (Fig. 4A-bGo). Lanes 4 and 6 show that 2.5-fold more of actin was detected in lanes 3 and 5 (Fig. 4A-bGo), and an identical amount of actin was detected in lanes 7 and 8.



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Figure 4. Expression of TR Isoforms in the Liver and Heart of TRßPV Mice

Nuclear extracts from liver (2 mg and 5 mg in lanes 3/5 and 4/6, respectively) or heart (5 mg, lanes 7 and 8) were immunoprecipitated using 15 µg of mAb C4 or MOPC according to Materials and Methods. The immunoprecipitated proteins were analyzed by Western blotting using {alpha}1–403 (1:2, 500 dilution) (A-a) or in (B): IgG-ß1 (5 µg/ml) or in (C): C91 (5 µg/ml) as described in Materials and Methods. Lanes 1 and 2 show the [35S]methionine labeled in vitro translated TR{alpha}1 and TRß1, respectively, as controls for the molecular markers of these two TRs (A-b). Nuclear extracts of the liver (10 µg; lane 2) and of the heart (100 µg; lanes 3 and 4) were loaded onto a 10% SDS polyacrylamide gel. After being transferred to PVDF membrane, actin was detected using antiactin antibody (0.2 µg/ml) as described in Materials and Methods.

 
To detect TRß1 in the liver of TRß+/+ mice, we stripped the blot and carried out Western blot analysis using IgG-ß1 that specifically recognizes TRß1 (11). As shown in lanes 3 and 4 of Fig. 4BGo, full-length (band a) and truncated TRß1 (band b) were detected in nuclear extracts in a concentration-dependent manner. Only a nonspecific band was detected when MOPC (an irrelevant antibody) was used (lanes 5 and 6, Fig. 4BGo).

To determine the relative expression of TR{alpha}1 and TRß1 in the liver, we used C91 that recognizes the common epitope at the C-terminal region (Fig. 1Go, A and B) (13) in Western blot analysis. As shown in lanes 3 and 4 of Fig. 4CGo, the full-length TRß1 (band a) and truncated TR{alpha}1 (band c) were well separated. Therefore band b in lanes 3 and 4, represent a mixture of the full-length TR{alpha}1 and the truncated TRß1. Unfortunately, we were unsuccessful in separating the full-length TR{alpha}1 from the truncated TRß1 after numerous attempts with different conditions. However, because the ratios of the full-length TR{alpha}1 and truncated TR{alpha}1 remained constant, we were able to calculate the relative expression of TRß1/TR{alpha}1 to be approximately 3, based on the intensities of the well-separated truncated TR{alpha}1 (band c, Fig. 4CGo) and TRß1 (band a, Fig. 4CGo). These results are consistent with previous findings using other methods in which the ratios of TRß1/TR{alpha}1 in the liver of rats (11) and mice (12) was determined to be approximately 4.

Loss of T3 Binding Activity in the Nuclear Extracts of Liver, But Not the Heart, of TRßPV Mice
To examine whether the expression of the PV gene in the liver and heart led to any loss of T3 binding activity, we prepared nuclear extracts from these two tissues and carried out competitive T3 binding assays (Fig. 5Go). Analyses of competitive binding data indicate that in the liver (Fig. 5AGo), the maximal T3 binding capacities were 382 ± 21, 160 ± 8, and 80 ± 9 fmol/mg protein for TRß+/+, TRßPV/+, and TRßPV/PV mice, respectively. These data indicate a reduction of 80% in the maximal binding capacity of the nuclear extracts of TRßPV/PV mice as compared with that in TRß+/+ mice (Fig. 5AGo; P < 0.05). Because it is known that only TRß1 and TR{alpha}1, but not TRß2, are expressed in the liver (16), these data would indicate that 4-fold more of TRß1 protein than TR{alpha}1 protein was expressed in the liver. There were no significant differences in the T3 binding affinities of the liver nuclear extracts of TRßPV/+ [dissociation constant (Kd) = 0.26 ± 0.03 nM] and TRßPV/PV (Kd = 0.59±0.11 nM) mice as compared with that of TRß+/+ mice (Kd = 0.24 ± 0.04 nM).



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Figure 5. Binding of T3 to the Nuclear Extracts of the Liver (A) and Heart (B) of Wild-Type and TRßPV Mice

Equal amounts of nuclear extract (heart, 100 µg; liver, 50 µg) were incubated with 0.4 nM of [125I]T3 in the absence or presence concentrations of unlabeled T3 as indicated (0.25, 0.5, 1, 2, 10, and 10,000 nM). The free and bound [125I]T3 were separated as described in Materials and Methods. Data are expressed as % of the [125I]T3 bound in the absence of unlabeled T3. The curves were fitted to the data, using Eq 1 as shown in Materials and Methods. Symbols for different strains of mice are as marked. Data are expressed as % of [125I]T3 bound specifically by extracts from the livers of wild-type mice, in the absence of competing unlabeled T3. C, Comparison of T3 binding capacity in the liver and heart of the wild-type and TRßPV mice.

 
In contrast to the liver, no significant differences were found in the maximal binding capacities of the nuclear extracts from the hearts of TRß+/+, TRßPV/+ and TRßPV/PV mice (15 ± 3.3, 8.1 ± 3.3 and 15.2 ± 11 fmol/mg protein, respectively). These data suggest that the contribution of TRß1 to the total T3 binding was relatively small, such that the mutation of both TRß gene alleles in TRßPV/PV mice had no discernable effect on the maximal binding capacity of the nuclear extracts in the heart. This notion is consistent with the findings from the Western analysis shown above in that the TRß1 protein in the heart is less than 10% of that in the liver. The maximal T3 binding capacity in the heart was only approximately 4% of that in the liver of TRß+/+ mice, which is consistent with the Western blot analysis shown in Fig. 4Go. However, the present study shows that the ratio of T3 binding capacity in the liver vs. in the heart (liver/heart = 25) differed significantly from that reported in the rats (liver/heart = 1.7; Ref. 11). Similar to that found in the liver, no significant changes were detected in the binding affinities of the nuclear extracts of the hearts of TRßPV/+ and TRßPV/PV mice as compared with that of TRß+/+ mice.

Competition of Mutant PV with w-TRs for Binding to TRE in the Liver
To understand how the expression of PV in the liver and heart affects the interaction of w-TR isoforms with TRE, we prepared nuclear extracts from these two tissues of TRß+/+, TRßPV/+, and TRßPV/PV mice and carried out EMSA. We chose to use Lys-TRE in EMSA because this TRE bind TRs with a high affinity so that the TR expressed in low abundance in the heart can be detected for comparison (8, 17). We also used the nuclear extracts from the liver of mice deficient in TR{alpha}1 (TR{alpha}1-/- mice) or TRß1 (TRß1-/- mice) (18) as controls for the binding of TRE to w-TRß1 or w-TR{alpha}1, respectively. Lanes 1–5 of Fig. 6AGo show the binding of Lys-TRE to the liver nuclear extracts of TR{alpha}1-/-, TRß-/-, TRß+/+, TRßPV/+ and TRßPV/PV 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 6. Binding of TR Isoforms and Mutant PV in the Liver Nuclear Extracts of Wild-Type or TRßPV Mice to Lys-TRE

Equal amounts of nuclear extracts (12 µg) from different strains of mice as marked were incubated with [32P]-labeled TRE-Lys in the presence (A, lanes 6–25; B, lanes 1–17) or absence (A, lanes 1–5) of anti-TR isoform specific antibodies as marked according to 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. 1Go, A and B) to specifically supershift TRE-bound TR isoforms. Lane 6 of Fig. 6AGo shows that in TR{alpha}1-/- mice, TRE-bound TRß1 was specifically supershifted by IgG-ß1; whereas no band with the same mobility was detected in mice deficient in TRß-/- (lane 7). Lanes 8–9 show that TRß1 was supershifted, but with decreasing intensities, which is consistent with the genotype in that only one allele of w-TRß is expressed in TRßPV/+ mice. However, the decreased intensity in the band in lane 9 was intriguing because the epitope of IgG-ß1 is located in the A/B domain that is common in TRß1 and PV proteins (Fig. 1BGo). These observations suggest that the C-terminal region of PV, that has a totally different sequence from that of the TRß1, could weaken the interaction of an epitope in the A/B domain of PV with IgG-ß1 than that in TRß1, suggesting that the C-terminal region of PV could affect the structure of A/B domain in the context of tertiary structure of the receptor. This notion is supported by the detection of a supershifted TRE-bound PV band in lane 10 that was weaker than that in lane 9. The band in lane 10 represented PV/IgG-ß1 complexes in TRßPV/PV mice without the contribution of TRß1 seen in TRßPV/+ mice (lane 9).

Lane 12 of Fig. 6AGo shows that TR{alpha}1 was specifically supershifted by {alpha}1–403 with faster mobility than that of TRß1/Ab complexes, which is consistent with the lower molecular weight of TR{alpha}1. In contrast, no supershifted band was detected in TR{alpha}1-/- mice as expected (lane 11). Lane 12 further shows that virtually all of the TRE-bound TR{alpha}1 was shifted because very little TRE-bound signal remained in the faster migration region (bands A and B) (also compared with lane 7). The supershifted TRE-bound TR{alpha}1 bands with similar mobility as that in lane 12 were also detected in TRß+/+, TRßPV/+ and TRßPV/PV mice (lanes 13–15, respectively). It is important to point out that the intensities of these supershifted TRE-bound TR{alpha}1 bands were similar to that in TRß1-/- mice (compare lanes 13–15 with lane 12). Furthermore, strong faster migrating bands A and B remained unsupershifted, which we postulate to be TRE-bound TRß1. To confirm that this was the case, both antibodies, IgG-ß1 and {alpha}1–403, were simultaneously used to supershift TRE-bound TRs. As shown in lanes 16–20, TRE-bound TR{alpha}1 and TRß1 were supershifted with well-separated mobility similar to that seen using single isoform-specific antibody. Lanes 21–25 were the negative controls to demonstrate the specificity in that when an irrelevant antibody, MOPC, was used in the supershifted experiments, no supershifted bands were detected. Taken together, these EMSA patterns shown in Fig. 6AGo clearly support the results from immunoprecipitation/Western blot analysis shown above, in that TRß1 is the major TR isoform in the liver and that in vivo, the extent of occupancy of TRE correlates with the abundance of TR isoforms.

To confirm that the supershifted bands detected in lanes 9, 10, 19, and 20 of Fig. 6AGo contained TRE-bound mutant PV, we used the anti-PV specific antibody T1 (13) in EMSA (Fig. 6BGo). Lanes 1, 2, and 3 of Fig. 6BGo show the supershifted TRE-bound receptor/Ab complexes detected in TRß+/+, TRßPV/+ and TRßPV/PV mice, respectively. When T1 was used, no T1-supershifted receptor was detected in TRß+/+ mice (lane 4, Fig. 6BGo) as expected in the wild-type mice. In TRßPV/+ mice (lane 5, Fig. 6BGo), two T1-supershifted bands with different mobility were detected (marked by * and {Delta}). The faster mobility band (*) migrated to the same position as the supershifted TRE-bound TR{alpha}1 (compared with the supershifted TRE-bound TR{alpha}1 in lanes 10–12, Fig. 6BGo). The slower mobility band ({Delta}) was at the same corresponding position as supershifted TRE-bound TRß1 (compared with the supershifted TRE-bound TRß1 in lanes 1–3). Thus, the more retarded T1-supershifted band represented TRß1/PV and PV/PV. The faster T1-supershifted band represented TR{alpha}1/PV (marked by *). To further support this conclusion, we used both IgG-ß1 and T1 simultaneously in EMSA. As shown in lane 8, a third supershifted band with the most retarded mobility ({diamondsuit}) was seen, representing the binding of both IgG-ß1 and T1 to the TRß1/PV heterodimer, the former bound to the epitope in the A/B domain of TRß1 and the latter bound to the C-terminal PV epitope (see Fig. 1Go, A and B). Lane 6 shows that in homozygous TRßPV/PV mice, the intensity of the T1-supershifted band ({Delta}) was increased, indicating the binding of T1 to PV/PV homodimers. When IgG-ß1 and T1 were both present, the most retarded band in lane 9 ({diamondsuit}) represented the binding of IgG-ß1 to the A/B domain and the binding of T1 to the C-terminal PV in PV/PV homodimers.

Figure 6BGo also shows that TR{alpha}1 forms a heterodimer with PV. T1 supershifted PV in the heterozygous mice to the same corresponding position (lane 5, marked by *) as that shown in lanes 10–12 in which TR{alpha}1 was supershifted by {alpha}1–403 alone. The intensity of the T1 supershifted band (*) in lane 6 was intensified in the TRßPV/PV mice as compared with in lane 5, indicating the binding of more PV/TR{alpha}1 to TRE. When both antibodies, T1 and {alpha}1–403, were used simultaneously in EMSA, the intensities of T1 supershifted PV/TR{alpha}1 bands in TRßPV/+ mice (lane 14; marked by *) and TRßPV/PV mice (lane 15; marked by *) were increased as compared with those in which either antibody was present alone (lanes 5 and 6 with T1; lanes 11 and 12 with {alpha}1–403). The increased intensity of the more retarded band in lane 15 (marked by {diamondsuit}) as compared with the more retarded band in lane 6 (marked by {Delta}), indicated that the more retarded band in lane 15 (marked by {diamondsuit}) represents a mixture of PV/PV and PV/TR{alpha}1. The latter is due to the simultaneous binding of antibody T1 to PV and antibody {alpha}1–403 to TR{alpha}1. To further confirm that this is the case, PV/TR{alpha}1 band was supershifted by using both antibodies {alpha}1–403 and IgG-ß1. As shown in lanes 16 and 17 of Fig. 6BGo, a more retarded band (marked by {diamondsuit}) was detected. The increased intensity of this more retarded band in lane 17 (marked by {diamondsuit}) as compared with the IgG-ß1-supeshifted band in lane 3 indicates that the more retarded band in lane 17 (marked by {diamondsuit}) represents a mixture of PV/PV and PV/TR{alpha}1. The latter is due to the simultaneous binding of antibody IgG-ß1 to the amino terminus of PV and antibody {alpha}1–403 to the C-terminus of TR{alpha}1. Taken together, these results provide direct evidence to indicate that in vivo, PV indeed competed with wild-type TRs for binding to TREs. In TRßPV/PV mice, PV competed more effectively with w-TR{alpha}1 for binding to TRE as PV/PV and PV/TR{alpha}1.

To understand whether PV also binds to TRE as a dimer with RXR, we used an antibody, 1D-12, which recognizes all RXR isoforms in the supershift experiments (Fig. 7Go) (19). 1D-12 recognizes the common regions in D/E domain of all RXR isoforms (19). Lanes 4, 5, and 6 of Fig. 7Go show that 1D-12 supershifted TRE-bound receptors in TRß+/+, TRßPV/+, TRßPV/PV mice, respectively. Because RXRs do not bind to Lys-TRE (7), the 1D-12-supershifted bands in lane 4 (TRß+/+ mice) represented TR{alpha}1/RXR and TRß1/RXR dimers; in lane 5, TR{alpha}1/RXR, TRß1/RXR and PV/RXR (TRßPV/+ mice); and in lane 6, TR{alpha}1/RXR and PV/RXR (TRßPV/PV mice). To confirm the formation of TRE-bound PV/RXR in TRßPV/+ and TRßPV/PV mice, we used both anti-PV (T1) and anti-RXR (1D-12) antibodies in EMSA. As shown in lane 11 (TRßPV/+ mice), a more retarded band (marked by {Delta}) than that in lane 8 supershifted by T1 alone and that in lane 5 supershifted by 1D-12 alone, was detected, indicating the formation of PV/RXR dimer. As expected, no T1-supershifted band was detected in lane 7, nor in lane 10 from the TRß+/+ mice. The formation of PV/RXR dimers was further confirmed by the increased intensity of PV/RXR dimer band in TRßPV/PV mice (band marked as {Delta} in lane 12 and compared with that in lane 11) and the concomitant reduction in the 1D-12 supershifted band (marked as {diamondsuit} and compared with that in lane 11). These results indicate that PV also competes with w-TRs for RXR in binding to TRE. In TRßPV/PV mice, this competition led to reduction in the binding of RXR/TR{alpha}1 to TRE.



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Figure 7. Competition of Binding of Mutant PV with w-TR Isoforms for Heterdimerization with RXR in Binding to Lys-TRE in the Liver Nuclear Extracts of TRßPV Mice

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 4–12) or absence (lanes 1–3) of anti-PV specific antibodies and anti-RXR antibodies as marked according to Materials and Methods [anti-RXR antibody, 1D-12: (1:100 dilution)/lane: T1: 2 µg/lane].

 
Ineffective Competition of Mutant PV with TR{alpha}1 for Binding to TRE in the Heart
A different DNA binding pattern emerged for the TRs in the heart (Fig. 8AGo). The binding of TRs from the hearts of TRß+/+, TRßPV/+, and TRßPVPV mice to TRE was weak (lanes 1–3, Fig. 8Go) even though an identical amount of nuclear extracts as that in the liver was used in the EMSA, as described above. This indicates that the abundance of TRs in the heart was substantially lower than that in the liver, which is consistent with the results of Western blot analyses shown above. Discernable, but weak, {alpha}1–403 supershifted TR{alpha}1 bands were detected in TRß+/+, TRßPV/+ and TRßPV/PV mice (lanes 4, 5, and 6, respectively, Fig. 8AGo). There were no significant differences in the intensities of the TRE-bound supershifted bands derived from the three genotypes, indicating that an identical abundance of TR{alpha}1 was expressed in the hearts of TRß+/+, TRßPV/+ and TRßPV/PV mice. However, under identical experimental conditions, neither IgG-ß1-supershifted TRß1 in TRß+/+, and TRßPV/+ mice (lanes 7 and 8, respectively), nor T1-supershifted PV in TRßPV/+ and TRßPV/PV mice (lanes 11 and 12, respectively) were detected, indicating that the abundance of TRß1 and PV was too low to be detected. These results further confirmed that TR{alpha}1 is the major TR isoform in the heart. More importantly, the expression of PV in the heart is too low to effectively compete with TR{alpha}1 for binding to TRE.



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Figure 8. Binding of Lys-TRE to the Nuclear Extracts of Heart and Liver of Wild-Type and TRßPV Mice

A, Binding of TR{alpha}1 in the heart nuclear extracts of wild-type or TRßPV 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 4–12) or absence (lanes 1–3) of anti-TR isoform and anti-PV-specific antibodies as marked according to Materials and Methods. B, Comparison of binding of w-TR isoforms in the heart (lanes 1–3) and liver (lanes 4–6) nuclear extract of wild-type mice. Equal amounts of nuclear extracts (12 µg) were incubated with [32P]-labeled TRE-Lys in the presence (lanes 2, 3, 5, and 6) or absence (lanes 1 and 4) of anti-TRß1 antibody (IgG-ß1: 2 µg in lanes 3 and 6) and anti-TR{alpha}1 antibody [{alpha}1–403: (1:100 dilution in lanes 2 and 5)] according to Materials and Methods.

 
Because the abundance of TR{alpha}1 in the heart was too low to be detected by the two-step Western blot analysis (see Fig. 4AGo), we could not determine the relative ratios of TR{alpha}1 in the liver and the heart. The detection of TRE-bound TR{alpha}1 by EMSA shown in lanes of 4–6 of Fig. 8AGo provided us with a tool to estimate the ratios of the abundance of TR{alpha}1 between the heart and the liver (Fig. 8BGo). In separate experiments, using identical amounts of nuclear extracts from the heart and the liver of TRß+/+ mice, we compared the intensities of the TRE-bound TR{alpha}1 (lanes 2 and 5) and TRß1 (lanes 3 and 6) after an extended period of exposure of the autoradiograms. Quantification of the intensities of the TRE-bound {alpha}1–403 supershifted bands in the liver (lane 5, Fig. 8BGo) and in the heart (lane 2, Fig. 8BGo) indicates a ratio of approximately 6 (liver:heart). However, no TRE-bound TRß1 supershifted by IgG-ß1 was discernable in the heart (lane 3, Fig. 8BGo), in spite of an extended period of exposure of the autoradiogram, indicating that the abundance of TRß1 in the heart was clearly substantially lower than that in the liver.

Interference with the Transcription of w-TRs by PV in TRßPV Mice Is Tissue Dependent
The findings from EMSA shown above predict that the T3-positively regulated target genes in the liver would be repressed in TRßPV mice due to an effective competition of PV with w-TRs for binding to TRE in the promoter of T3 target genes. In contrast, the regulation pattern of T3 target genes in the heart would be normal due to the lack of competition of w-TR with PV and the increased circulating levels of thyroid hormones in TRßPV mice (9). To find out whether this was the case, we analyzed the expression of T3-target genes in the liver (Fig. 9Go) and heart (Fig. 10Go) of TRßPV mice by Northern blot analysis. The expression of ME, S14, and D1 in the liver is known to be positively regulated by T3. However, instead of being activated by the increased thyroid hormones, these genes were repressed as shown in Fig. 9Go, A–C. The expression of ME, S14, and D1 was repressed 40%, 60%, and 50%, respectively, in TRßPV/+ mice, indicating the inhibition of transcriptional regulation of the w-TRs by PV. The expression of ME, S14, and D1 was further repressed (70–95%) in the TRßPV/PV mice, indicating that the extent of the repression of these genes was enhanced when the abundance of PV protein was increased in TRßPV/PV mice, leading to more effective competition with TR{alpha}1 for TREs (also see Figs. 6Go and 8Go).



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Figure 9. The Expression of T3-Positively Regulated Genes in the Liver of TRßPV Mice Was Repressed

Total RNA was isolated from the liver of mice (three for each group of 8-wk-old male mice). The intensities of the bands were quantified using a Molecular Dynamics, Inc. PhosphorImager. The levels of the expression of the malic enzyme (A), S14 (B) and DI (C) were normalized using GAPDH mRNA. Data are expressed as mean ± SD. D, n = 3. The differences in the expression of these genes between TRß+/+ and TRßPV/+ mice or between TRß+/+ and TRßPV/PV mice are significant (P < 0.05).

 


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Figure 10. The Expression of {alpha}- and ß-MHC in the Heart of TRßPV Mice Was Altered

Total RNA was isolated from the heart of mice (three for each group of 8-wk-old male mice). The intensities of the bands were quantified using a Molecular Dynamics, Inc. PhosphorImager. The levels of the expression of these two genes were normalized using GAPDH mRNA. The differences in the expression of {alpha}-MHC between TRß+/+ and TRßPV/PV mice (A) and the differences in the expression of ß-MHC between TRß+/+ and TRßPV/+ or between TRß+/+ and TRßPV/PV mice (B) are significant (P < 0.04).

 
A very different expression pattern of T3-target genes emerged in the heart of TRßPV mice (Fig. 10Go). {alpha}- and ß-myosin heavy chain genes ({alpha}-MHC; ß-MHC) are positively and negatively regulated by T3, respectively, in the heart (20). Figure 10Go shows that, in contrast to the T3-positively regulated genes in the liver, the expression of {alpha}-MHC in the heart of TRßPV/+ mice was slightly increased (~15%), reflecting the activated response to the moderate increase in the circulating TT3 and TT4 in TRßPV/+ mice. A higher activation (1.8-fold) in the expression of {alpha}-MHC was detected in the heart of TRßPV/PV mice (Fig. 10AGo), responding to a much higher circulating total T4 (15-fold increase) and total T3 (9-fold increase) in TRßPV/PV mice. Figure 10BGo further shows that the expression of ß-MHC was repressed approximately 65% in the heart of TRßPV/+ and TRßPV/PV mice, indicating the response of this T3-negatively targeted gene to the higher circulating levels of thyroid hormones. Taken together, the appropriate response of these two cardiac genes to thyroid hormones indicates that PV, expressed at a significantly lower abundance, as compared with TR{alpha}1, cannot effectively compete with TR{alpha}1 for binding to TRE. Thus, no inhibition of the transcriptional activity of TR{alpha}1 was observed in the heart.

Interference with the Transactivation Activity of w-TRs Is Reversed by Increasing Expression of TR{alpha}1
The findings from the DNA binding and expression patterns of T3-positively regulated target genes in the liver and heart suggest that the in vivo action of PV protein depends on its abundance and the relative abundance of TRß1 and TR{alpha}1 proteins in the T3 target tissues. As shown above, TRß1 is the major TR isoform in the liver. In line with the expression of TRß (9), a high level of PV protein was present in the liver, leading to effective competition with the w-TRß1 and w-TR{alpha}1 for binding to TRE and resulting in the repression of T3 target genes. In contrast, TR{alpha}1 is the predominant isoform and no PV was detectable in the heart. This substantially lower expression level of PV, as compared with TR{alpha}1, was not sufficient for its competition with TR{alpha}1 for binding to TRE, resulting in no inhibition of the transcription activity of TR{alpha}1. Furthermore, because TRßPV mice have high circulating levels of thyroid hormone, the T3 target genes in the heart respond appropriately by either activation or repression depending on the type of T3 target genes.

To provide evidence to further support this notion, we turned to the use of the {alpha}-MHC-TK-CAT reporter system (21). To mimic the distribution of TRs in the heart, we transfected TRß1 and PV expression plasmids in the presence of increasing amounts of TR{alpha}1 expression plasmid in CV1 cells and evaluated the repression of {alpha}-MHC-TK-CAT reporter activity (21) by PV at each condition (Fig. 11Go). Bars 2 and 1 of Fig. 11Go show the T3-dependent activation of {alpha}-MHC-TK-CAT activity mediated by TRß1. Bars 4 and 3 show the T3-dependent activation of {alpha}-MHC-TK-CAT activity mediated by TR{alpha}1. Bar 5 shows that PV lacked T3-dependent transactivation activity (compare with bar 1 or 3). However, as shown in bar 6, cotransfection of PV expression vector repressed the TRß1-mediated T3-dependent transactivation (bar 6 vs. bar 2). This repression, however, was abrogated by the cotransfection of increasing amounts of TR{alpha}1 expression plasmid as shown in bars 7 and 8 (the TR{alpha}1:TRß1 ratio was 1 and 5 in bars 7 and 8, respectively). These results support the competition model in that an increasing expression level of TR{alpha}1 can effectively compete with PV for binding to TRE, thereby reversing the inhibition of PV on the transcriptional activity of TR{alpha}1.



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Figure 11. Abrogation of the Dominant Activity of PV on TRß1 by Increasing the Expression of TR{alpha}1

Transactivation activity: CV1 cells (6 x 105 cells/well) were transfected with p{alpha}-MHC-thymidine kinase-chloramphenicol acetyltransferase (p{alpha}-MHC-TK-CAT) (0.4 µg) and TRß1 expression plasmid (pCLC51; 0.2 µg) or TR{alpha}1 expression plasmid (pCLC61; 0.2 or 1 µg) in the absence or presence of the expression plasmid of PV (pCLC51PV; 0.2 µg) as indicated. After 48 h, the CAT activity in triplicates was determined using CAT ELISA Kit (Roche Diagnostics GmbH) according to the manufacturer’s instructions. The data are expressed as mean ± SD (n = 3).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The availability of TRßPV mice has made it possible to study the molecular mechanisms by which a mutant TRß exerts its dominant negative effect in vivo. Using a battery of TR isoform-specific antibodies to specifically interact with the endogenous w-TRs and mutant PV in the liver, the present work clearly shows that PV competed with w-TRs for binding to TRE not only as a heterodimeric partner with w-TRs, but also with RXR in vivo. This competition is clearly more effective in TRßPV/PV mice than in TRßPV/+ mice (Fig. 6BGo). The present study provides the direct in vivo evidence to support the model previously proposed for the molecular mechanisms of dominant negative action of mutant TRß based on in vitro studies. Meier et al. (7) and others (6) proposed that competition of mutant TRß with w-TR for binding to TRE (as inactive mutant/w-TR and mutant/mutant dimers) and/or the depletion of auxiliary factors, such as RXR, could account for the dominant negative action of mutant TRß. Our data clearly indicate that, in vivo, PV could act to affect the transcriptional activity of w-TR via these interrelated mechanisms. At present, due to the limitations of the resolving capacity of EMSA and the enormous complexity of multireceptor interactions and competitions of an array of dimers for binding to TRE in vivo, it is impossible to determine which one of these mechanisms plays a predominant role. Furthermore, our data could not exclude the contributions of other mechanisms that are yet to be identified and analyzed in future studies.

The demonstration of the critical role of the differential abundance of TR isoform proteins in dictating the dominant negative activity has gained insights into the molecular basis of the variability in tissue responsiveness in RTH patients. Two distinct T3-target gene response patterns in the liver and heart of TRßPV mice were detected. In the model presented in Fig. 12Go, we designate the liver as a type I tissue to represent the tissues that are resistant to thyroid hormone (Fig. 12AGo) as evidenced by the inhibition of the expression of T3-positively regulated target genes. We designate the heart as a type II tissue to represent tissues that could show signs of thyrotoxicosis as evidenced by the activation of the cardiac genes in response to the high circulating thyroid hormone levels (Fig. 12BGo). In the liver (a type I tissue), the abundance of TRß protein is higher than in TR{alpha}1 protein. The abundance of PV is in line with that of TRß as they utilize the same promoter as evidenced by the identical expression of their mRNAs (9). As schematically illustrated in Fig. 12AGo, in TRßPV/+ mice (A-a), an equal number of mutant receptors are competing with the w-TRß and also with w-TR{alpha}1 for binding to TRE of the T3-target genes. A weak inhibition of the transcriptional activity of w-TRs is expected because there are fewer mutant receptors as compared with the sum of w-TRß and w-TR{alpha}1. This notion is consistent with the mild phenotypic expression observed in most RTH patients (4) and TRßPV mice (9). In TRßPV/PV mice (A-b), the level of the mutant receptors is now increased 2-fold. The mutant receptors can now more effectively compete with a lower number of TR{alpha}1 for binding to TRE, leading to a stronger repression of the transcriptional activity of w-TR{alpha}1. This notion is supported by the extent of abnormal expression patterns of the T3-target genes (S14, ME, and D1) in the liver (see Fig. 9Go) in that more severe repressions of T3-positively regulated genes were detected in spite of a higher level of circulating thyroid hormones.



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Figure 12. Differential Expression of TR Isoforms in Tissues Leads to Different Responsiveness in RTH

A, In type I tissues, predominant expression of TRß results in resistance to thyroid hormones. B, In type II tissues, predominant expression of TR{alpha}1 leads to thyrotoxicosis. The relative thickness of the arrows schematically represents the magnitude of transcriptional response of the target genes.

 
Another example of type I tissue could be the pituitary, even though it was not possible to obtain sufficient nuclear extracts from the pituitary due to its extremely small size (the number of mice needed is prohibitive). However, based on in vitro studies and analyses of the phenotypes of TR isoform knockouts (7, 22), TRß most likely is the major isoform in the pituitary that is expected to fall into the class of type I tissue (Fig. 12Go). Indeed, the pituitary of RTH patients is resistant to the action of thyroid hormone exhibited by inappropriately normal or higher levels of thyrotropin (4) in the face of a high level of circulating thyroid hormone. This is further supported by the abnormal gene expression patterns in the pituitary of TRßPV mice in which both the {alpha}- and ß-subunits of thyrotropin are up-regulated and the thyrotropin-secreting cells are increased (9).

In the heart (a type II tissue), a very different picture emerges (Fig. 12BGo). In TRßPV/+ mice (shown in B-a), we have shown that abundance of TRß protein is significantly lower than that of TR{alpha}1 protein. Therefore, fewer mutant receptors are available to compete with w-TRß and with a higher number of TR{alpha}1 for binding to TRE. In TRßPV/PV mice (shown in B-b), even though the mutant receptors are increased 2-fold, they are still low in number as compared with TR{alpha}1 so that its binding to TRE is less favorable than TR{alpha}1. Because TRßPV/+ and TRßPV/PV mice have a higher level of circulating thyroid hormones than the wild-type mice (9), the T3-positively regulated genes are expected to be activated and the T3-negatively regulated genes to be down-regulated in the heart. Indeed, we found that this is the case in that the expression of {alpha}-cardiac MHC is activated and the expression of ß-cardiac MHC is repressed in TRßPV/+ and TRßPV/PV mice (see Fig. 10Go, A and B). Based on the model shown in Fig. 12Go, the expression of the TRß gene (in relation to the TR{alpha} gene), which determines the abundance of a TRß mutant, would dictate the phenotypic expression of RTH in a tissue.

The model in Fig. 12Go illustrates that competition of mutants with w-TRs in binding to T3-target genes is a critical step in the action of TR mutants. However, it is important to point out that the abnormal regulation patterns of T3-target genes by mutant TR in type I tissues are subject to fine tuning by other factors including the type of TREs present in the target genes, additional upstream transcription factors and TR coregulatory proteins. Thus, depending on the type of T3-target genes, the extent of abnormal regulation by mutant TR will vary. This is clearly demonstrated by the findings that in the liver, the extent of abnormal regulation of S14 gene is different from that of D1 (Fig. 9Go). In the type II tissues, the extent of potentiation of T3-target genes will again be subject to further modulation by other factors. This is apparent in Fig. 10Go in that the extent of the activation of the {alpha}-MHC is less pronounced as compared with the repression of ß-MHC.

Recent studies using mice deficient in TR{alpha} and/or TRß genes indicate that TR isoforms have both specific and common functions (18). The TR isoform-specific gene regulation in a tissue may be conferred by a difference in the expression levels of TR{alpha}1 and TRß proteins. The present study indicates that the differential expression of TR{alpha}1 and TRß proteins in tissues dictates the extent of the dominant negative action of mutant TRß in vivo, leading to clinical consequences. Therefore, the differential expression of TR isoforms dictates not only the action of w-TR in health but also in disease.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Strains of Mice
TRßPV mice were generated and bred as described by Kaneshige et al. (9). Mice deficient in TR{alpha}1 and TRß were obtained from B. Vennstrom and D. Forrest (18).

Preparation of Nuclear Extracts
Nuclear extracts were prepared by modification of the methods of Frain et al. (23) and Chamba et al. (24). Briefly, livers or hearts 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 1,500 x g for 15 min. The washed nuclei were resuspended in buffer C (buffer A without sucrose) and centrifuged at 1,500 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, and Complete Mini EDTA-free and KCl was 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.

Preparation of Monoclonal Anti-PV Antibodies (mAb 302)
Mice were immunized with keyhole limpet hemocyanin conjugated-peptide with the C-terminal sequence of PV shown in Fig. 1AGo (14, 25). The peptide was purified by high pressure liquid chromatography and the sequence of the PV was confirmed by amino acid analysis. Spleen cells from the immune mice were fused with P3X63 Ag 8653 myeloma cells by the method described in (25). The hybridimas were screened by immunoprecipitation with [35S]-methionine-labeled in vitro translated PV protein and subsequently confirmed by Western blot analysis using the cell lysates transfected with the expression vector of PV. The hybridomas secreting anti-PV antibodies were injected into mice to obtain ascites, which were purified by ammonium sulfate precipitates.

Determination of the Abundance of TR Proteins in Tissues by Immunoprecipitation and Western Blot Analysis
To determine the abundance of w-TRß or PV protein, nuclear extracts (1–10 mg) were treated with protein G agarose suspension (30–50 µl) (Roche Molecular Biochemicals) which was prebound to antimouse IgG rabbit antibody overnight. After centrifugation at 12,000 x g for 10 min at 4 C, the supernatant was immunoprecipitated with anti-TRß1, IgG-ß1, (10 µg; a generous gift from Dr. C. Mariash, University of Minnesota, Minneapolis, MN) (11) or the monoclonal anti-PV antibody, mAb 302 (10 µg) and protein G agarose suspension (30–50 µl) overnight. After washing, the agarose pellet was boiled in 2x sodium dodecyl sulfate (SDS) sample buffer for 5 min and centrifuged at 12,000 x g for 20 sec; the supernatant was separated by 10% SDS-PAGE. After the proteins were transferred to polyvinylidene difluoride (PVDF) membrane, Western blot analysis was performed using mAbC4 (2 µg/ml) (13), anti-PV antibodies, T1 (0.5 µg/ml) or MOPC (2 µg/ml; Sigma, St. Louis, MO) as control. To control the loading of proteins, nuclear extracts (20 and 50 µg) were loaded onto the gel. After electrophoresis and transfer to the PVDF membrane, monoclonal antiactin antibody (0.2 µg/ml; Roche Molecular Biochemicals) was used to detect actin.

To detect w-TR{alpha}1 protein, nuclear extracts (1–10 mg) were incubated overnight with 15 µg of mAbC4 or MOPC (negative control). After 40 µl of protein G agarose suspension (Roche Molecular Biochemicals) were added and incubated for 3 h, the agarose beads were washed, the pellet was boiled in 2x SDS sample buffer for 5 min and centrifuged at 12,000 x g for 20 sec, and the supernatant was separated by 10% SDS-PAGE. After transferring the proteins to PVDF membrane, Western blot analysis was performed using anti-TR{alpha}1 antibody (1:2000; Affinity BioReagents, Inc., Golden, CO) (15) or polyclonal anti-TR antibodies, C91 (5 µg/ml) (14), and antirabbit IgG F(ab')2 fragment (1:5,000 dilution; Amersham Pharmacia Biotech, Piscataway, NJ).

Binding of T3 to TRs in the Nuclear Extracts of Liver and Heart of Mice
Nuclear extracts (100 µg) were incubated with 0.4 nM of (3'-125I)T3 in the presence of increasing concentrations of unlabeled T3. The TR-bound (3'-125I)T3 was separated from the free as described by Lin et al. (26). The binding data were analyzed by using equation I based on direct competition between (3'-125I)T3 and the unlabeled T3 for a single site on the receptor. The concentration of radioactive complex is given by the equation:

where [R]o is the total concentration of receptor, [h] and [c] are the concentrations of (3'-125I)T3 and the unlabeled T3, respectively, and Kd is the dissociation constant of the T3-receptor complex. The data were fitted directly to equation I using the PC-MLAB program (Civilized Software, Inc., Bethesda, MD), to evaluate Kd and [R]o. The fitted curves are shown in Fig. 5Go, A and B.

EMSA
The double-stranded oligonucleotide containing the Lys-TRE was labeled with [{alpha}-32P]deoxy-CTP similarly as described by Zhu et al. (27). About 0.2 ng of probe (3–5 x 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 carried out at room temperature for 30 min and 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 isolated from the liver and heart of TRßPV using Trizol Reagent (Life Technologies, Inc., Gaithersburg, MD). Total RNA (5–10 µg) was analyzed by electrophoresis and transferred onto membranes (Hybond-N+, Amersham Pharmacia Biotech, Arlington Heights, IL), which were hybridized with appropriate probes. cDNA for ME, spot 14, and type 1 deiodinase (D1), which were labeled with [{alpha}-32P]deoxy-CTP using a random 9-oligomer primer protocol. The probes for {alpha}-MHC and ß-MHC were {alpha}- or ß-MHC-specific 40-oligomers which were labeled as described by Gloss et al. (20). For quantification, the intensities of the mRNA bands were normalized against the intensities of GAPDH mRNA (n = 3). The blots were stripped and rehybridized with a [32P]-labeled GAPDH cDNA. Quantification was performed using the Molecular Dynamics, Inc. PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Construction of the Mammalian Expression Vector of Mutant PV
The cDNA encoding human mutant PV was released by treating the pSV2-PV (28) with EcoRI and XbaI, which was then cloned into the same sites of pcDNA3.1+ (Invitrogen, Carlsbad, CA) to give the intermediate plasmid pcDNA3.1+PV. pcDNA3.1+PV was digested with XbaI, which was then filled in to give blunt-ended plasmid with Klenow fragment. A 166-bp fragment containing the PV mutation site at the 3' end of the PV cDNA was released from the blunt-ended pcDNA3.1+PV using BglII. This fragment was then ligated onto pCLC51, a mammalian expression vector for the human TRß1 (26), after it was treated with NotI and blunt-ended similarly using Klenow fragment. Subsequently, it was digested with BglII to release the fragment at the 3' end of the TRß1 cDNA. The ligation of the BglII fragment containing the PV mutation yielded the expression vector for PV mutant, which was designated as pCLC51PV. The sequence of pCLC51PV was confirmed by DNA sequencing.

Transient Transfection Assay
Transient transfection experiments were carried out using CV1 cells as described by Lin et al. (29). Briefly, cells were transfected with plasmids containing the {alpha}-MHC TK-CAT (0.4 µg) and the expression plasmid for TRß1 (pCLC51; 0.2 µg) and/or the expression plasmid for TR{alpha}1 (pCLC61; 0.2 or 1 µg) (23) in the absence or presence of the expression plasmid of PV mutant (pCLC51PV; 0.2 µg). Five hours after transfection, cells were incubated in thyroid hormone deficient medium (Td medium). Twenty hours after transfection, T3 (100 nM) were added and incubated for an additional 24 h. Cells were lyzed and the CAT activity was determined using CAT ELISA (Roche Diagnostics GmbH, Mannheim, Germany). The values were normalized against the protein concentrations which were determined by the BCA protein assay kit (Pierce Chemical Co., Bradford, IL).


    ACKNOWLEDGMENTS
 
We thank Dr. I. Flink for the {alpha}-MHC TK-CAT reporter; Dr. C. Marish for the anti-TRß1 antibody IgG-ß1; Dr. P. Chambon for the anti-RXR antibody and 1D-12; and Li Zhao for the purification of T1, mAbC4, and mAb 302.


    FOOTNOTES
 
Abbreviations: CAT, Chloramphenicol acetyltransferase; D1, deiodinase 1; DTT, dithiothreitol; Kd, dissociation constant; mAb, monoclonal antibody; ME, malic enzyme; MHC, myosin heavy chain; PVDF, polyvinylidene difluoride; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; S14, spot 14; SDS, sodium dodecyl sulfate; TK, thymidine kinase; TRß, thyroid hormone receptor ß gene; TRE, thyroid hormone response elements; w-TR, wild-type TRs.

Received for publication February 22, 2002. Accepted for publication May 23, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Cheng SY 2000 Multiple mechanisms for regulation of the transcriptional activity of thyroid hormone receptors. Rev Endocr Metab Disorders 1:9–18[CrossRef][Medline]
  2. Williams GR 2000 Cloning and characterization of two novel thyroid hormone receptor ß isoforms. Mol Cell Biol 20:8329–8342[Abstract/Free Full Text]
  3. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  4. Weiss RE, Refetoff S 2000 Resistance to thyroid hormone. Rev Endocr Metab Disorders 1:97–108[CrossRef][Medline]
  5. Ono S, Schwartz ID, Mueller OT, Root AW, Usala SJ, Bercu BB 1991 Homozygosity for a dominant negative thyroid hormone receptor gene responsible for generalized resistance to thyroid hormone. J Clin Endocrinol Metab 73:990–994[Abstract]
  6. Yen PM, Chin WW 1994 Molecular mechanisms of dominant negative activity by nuclear hormone receptors. Mol Endocrinol 8:1450–1454[Medline]
  7. Meier CA, Parkison C, Chen A, Ashizawa K, Muchmore P, Meier-Heusler SC, Cheng SY, Weintraub BD 1993 Interaction of human ß1 thyroid hormone receptor and its mutants with DNA and RXR. T3 response element-dependent dominant negative potency. J Clin Invest 92:1986–1993[Medline]
  8. Zhu XG, Yu CL, McPhie P, Wong R, Cheng SY 1996 Understanding the molecular mechanism of dominant negative action of mutant thyroid hormone ß1 receptors: the important role of the wild-type/mutant receptor heterodimer. Endocrinology 137:712–721[Abstract]
  9. Kaneshige M, Kaneshige K, Zhu XG, Dace A, Garrett L, Carter TA, Kazlauskaite R, Pankratz DG, Wynshaw-Boris A, Weintraub B, Billingham MC, Barlow C, Cheng SY 2000 Mice with a targeted mutation in the thyroid hormone ß receptor gene exhibit impaired growth and resistance to thyroid hormone. Proc Natl Acad Sci USA 97:13209–13214[Abstract/Free Full Text]
  10. Parrilla RA, Mixson AJ, McPherson JA, McClaskey JH, Weintraub BD 1991 Characterization of seven novel mutations of the c-erbA ß gene in unrelated kindreds with generalized thyroid hormone resistance. Evidence for two "hot spot" regions of the ligand binding domain. J Clin Invest 88:2123–2130[Medline]
  11. Schwartz HL, Strait KA, Ling NC, Oppenheimer JH 1992 Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity. J Biol Chem 267:11794–11799[Abstract/Free Full Text]
  12. Weiss RE, Murata Y, Cua K, Hayashi Y, Seo H, Refetoff S 1998 Thyroid hormone action on liver, heart, and energy expenditure in thyroid hormone receptor ß-deficient mice. Endocrinology 139:4945–4952[Abstract/Free Full Text]
  13. Bhat MK, McPhie P, Ting YT, Zhu XG, Cheng SY 1995 The structure of the carboxyl-terminal region of thyroid hormone nuclear receptor and its role in hormone-dependent intermolecular interactions. Biochemistry 34:10591–10599[Medline]
  14. Fukuda T, Willingham MC, Cheng SY 1988 Antipeptide antibodies recognize c-Erb A and a related protein in human A431 carcinoma cells. Endocrinology 123:2646–2652[Abstract]
  15. Falcone M, Miyamoto T, Fierro-Renoy F, Macchia E, DeGroot LJ 1992 Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform. Endocrinology 131:2419–2429[Abstract]
  16. Yen PM 2001 Physiological and molecular basis of thyroid hormone action. Physiol Rev 81:1097–1142[Abstract/Free Full Text]
  17. Zhu XG, McPhie P, Cheng SY 1997 Differential sensitivity of thyroid hormone receptor isoform homodimers and mutant heterodimers to hormone-induced dissociation from DNA: its role in dominant negative action. Endocrinology 138:1456–1463[Abstract/Free Full Text]
  18. Forrest D, Vennstrom B 2000 Functions of thyroid hormone receptors in mice. Thyroid 10:41–52[Medline]
  19. Rochette-Egly C, Lutz Y, Pfister V, Heyberger S, Scheuer I, Chambon P, Gaub MP 1994 Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies. Biochem Biophys Res Commun 204:525–536[CrossRef][Medline]
  20. Gloss B, Sayen MR, Trost SU, Bluhm WF, Meyer M, Swanson EA, Usala SJ, Dillmann WH 1999 Altered cardiac phenotype in transgenic mice carrying the {Delta}337 threonine thyroid hormone receptor ß mutant derived from the S family. Endocrinology 140:897–902[Abstract/Free Full Text]
  21. Flink IL, Morkin E 1990 Interaction of thyroid hormone receptors with strong and weak cis-acting elements in the human {alpha}-myosin heavy chain gene promoter. J Biol Chem 265:11233–11237[Abstract/Free Full Text]
  22. Motomura K, Brent GA 1998 Mechanisms of thyroid hormone action: implications for the clinical manifestation of thyrotoxicosis. Endocrinol Metab Clin North Am 27:1–23[Medline]
  23. Frain M, Swart G, Monaci P, Nicosia A, Stampfli S, Frank R, Cortese R 1989 The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain. Cell 59:145–157[Medline]
  24. Chamba A, Neuberger J, Strain A, Hopkins J, Sheppard MC, Franklyn JA 1996 Expression and function of thyroid hormone receptor variants in normal and chronically diseased human liver. J Clin Endocrinol Metab 81:360–367[Abstract]
  25. Lin KH, Willingham MC, Liang CM, Cheng SY 1991 Intracellular distribution of the endogenous and transfected ß form of thyroid hormone nuclear receptor visualized by the use of domain-specific monoclonal antibodies. Endocrinology 128:2601–2609[Abstract]
  26. Lin KH, Fukuda T, Cheng SY 1990 Hormone and DNA binding activity of a purified thyroid hormone nuclear receptor expressed in Escherichia coli. J Biol Chem 265:5161–5165[Abstract/Free Full Text]
  27. Zhu XG, McPhie P, Lin KH, Cheng SY 1997 The differential hormone-dependent transcriptional activity of thyroid hormone receptor isoforms is mediated by interplay of their domains. J Biol Chem 272:9048–9054[Abstract/Free Full Text]
  28. Meier CA, Dickstein BM, Ashizawa K, McClaskey JH, Muchmore P, Ransom SC, Menke JB, Hao EH, Usala SJ, Bercu BB 1992 Variable transcriptional activity and ligand binding of mutant ß1 3, 5, 3'-triiodothyronine receptors from four families with generalized resistance to thyroid hormone. Mol Endocrinol 6:248–258[Abstract]
  29. Lin KH, Ashizawa K, Cheng SY 1992 Phosphorylation stimulates the transcriptional activity of the human ß1 thyroid hormone nuclear receptor. Proc Natl Acad Sci USA 89:7737–7741[Abstract]