We have successfully inactivated the β2 isoform of the TR. Our strategy was based on the fact that TRβ1 and TRβ2 differ only in their NH2
-termini and that these are encoded by distinct exons (5
). The relative location of the exons encoding the TRβ1 and TRβ2 NH2
-termini are not known. It is also likely that in addition to alternate exon use, distinct promoters drive the expression of these 2 TR isoforms (6
). It is clear from our data that disruption of the TRβ2-specific exon does not interfere with the splicing of the TRβ1-specific exons and the shared common exons. The selective targeting of TRβ2 also did not affect the spatial expression of the TRβ1 gene product. Although we did not examine the temporal expression of TRβ1 in this study, our data strongly suggest that the regulatory elements governing TRβ1 expression are not located within the TRβ2-specific exon, nor in the first 600 bp of the adjacent intron that was also inactivated by our targeting strategy. This targeting strategy ensured that the entire coding sequence of the unique TRβ2 NH2
-terminus was deleted. In fact, we were unable to detect by RT-PCR the existence of a truncated NH2
-terminal fragment that could potentially act as a dominant inhibitor. Therefore, these mice enabled us to define the physiological role of TRβ2 in the regulation of the hypothalamic-pituitary-thyroid axis, and provided novel insight into differential roles of TRβ1 and TRβ2 in the regulation of auditory function.
The selective disruption of TRβ2 confirms the important role that this isoform plays in the regulation of the hypothalamic-pituitary-thyroid axis. The 2- to 3-fold increase in thyroid hormone concentration seen in mice lacking TRβ2 is similar to that reported in mice lacking the entire TRβ locus (21
). The absolute hormone concentrations are not comparable, because our control values are lower than those reported by Forrest et al. (21
). Nevertheless, it appears that the absence of TRβ2 can account in large part for the hormonal abnormalities described in TRβ-null mice. The residual TRβ1 is therefore unable to compensate for the absence of TRβ2. This may reflect qualitative differences in the ability of TRβ1 to mediate the negative regulation of TSH by thyroid hormone. This possibility is supported by a large body of in vitro data that suggest that TRβ2 is a more potent negative regulator of the TSH-subunit genes than is TRβ1 (16
). However, the lack of TRβ2 does not completely abrogate the responsiveness of the thyrotroph to thyroid hormone, as evidenced by the partial suppression of TSHβ mRNA after 3 weeks of T3
treatment. The interpretation of the decline in T4
levels is more complex. The fall in T4
not only reflects impaired TSH suppression, but also reflects the ability of T3
to increase the peripheral clearance of T4
). This is one possible explanation for the similarity in the slopes of the decline in T4
in WT and TRβ2-null mice. The important difference, though, is the presence of measurable T4
in the TRβ2-null animals at a time when T4
levels are undetectable in WT mice. We believe that the persistence of T4
in the TRβ2-null mice after T3
treatment is due to persistence of TSH.
The partial suppression of TSH in response to T3
administration suggests that the residual TRβ1 and TRα1 are capable of mediating T3
-induced repression of the TSH-subunit genes. The recent reports of markedly elevated thyroid hormone concentrations in mice lacking both TRα and TRβ isoforms support this (35
). However, our data indicate that TRβ2 is required to mediate complete suppression of the axis by thyroid hormone. Whether or not TRβ2 alone can mediate all of the negative regulation of TSH production by thyroid hormone in vivo will only come from studies in mice with ablation of TRβ1 and TRα, but with preserved expression of TRβ2. The demonstration that the central resistance to thyroid hormone occurring in TRβ2 KO mice is due in part to impaired downregulation of TSHβ gene expression by T3
does not preclude the possibility of impaired regulation at the level of the TRH neuron, given the expression of TRβ2 in the T3
-responsive TRH neurons of the paraventricular hypothalamus (PVN) (9
). Our model now provides an important resource with which to analyze the role of TRβ2 in the regulation of TRH expression in the PVN over a wide range of thyroid hormone concentrations.
An important new finding from this study is the demonstration in vivo that TRβ2 is a critical mediator of the ligand-independent activation of TSHβ gene expression. In transfection experiments, we demonstrated previously that TRβ2 is a much more potent mediator of ligand-independent activation of the TSH-subunit and TRH genes than either TRβ1 or TRα1 (17
). A potential mechanism for the differential activation capabilities of the TR isoforms on negatively regulated genes may reside in differential interactions with accessory proteins such as corepressors and coactivators. We have demonstrated previously that the ability of TRβ2 to mediate ligand-independent activation is independent of its interactions with N-CoR (20
). In contrast, N-CoR binding limits the ligand-independent activation capability of TRβ1 and TRα1, and cotransfection of a potent dominant inhibitor of N-CoR converts these isoforms into strong mediators of ligand-independent activation (20
). The molecular mediators of the ligand-independent activation of the TSHβ gene by TRs are currently unknown. It is likely that the enhanced potency of TRβ2 may reside in enhanced activation via its interaction with coactivator molecules.
Although our findings of impaired in vivo ligand-independent activation of the TSHβ gene strongly support earlier in vitro evidence that TRβ2 is the important mediator of ligand-independent activation, 2 additional possibilities warrant discussion. First, because TRβ2 is the most abundant TR isoform in the pituitary, loss of TRβ2 reduces the total number of pituitary TRs. Thus, the observed phenotype could simply represent the result of a net reduction in pituitary TR content. To ultimately prove that this defect is specific to the TRβ2 isoform, mice that selectively lack TRβ1 and TRα1 while retaining TRβ2 expression would need to be studied. If TRβ2 is the key mediator of ligand-independent activation of TSHβ in vivo, then such animals would be expected to exhibit no defects in the TSH response to hypothyroidism. Second, it could be argued that the impaired induction of TSHβ expression by hypothyroidism in the TRβ2-null mice may simply be due to the fact that basal TSHβ gene expression is already increased. We believe that this is unlikely because of our observations in transgenic mice with pituitary expression of a mutant TR transgene (37
). Like the TRβ2-null mice, these animals exhibit pituitary resistance with a similar increase in basal TSH concentration. These animals increase their TSH concentrations 40-fold above baseline in response to hypothyroidism, indicating that despite higher basal TSH levels, thyrotroph responsiveness is retained. It is of interest that these transgenic mice with pituitary expression of a mutant TR express 40% less TSHβ mRNA than controls with similar degrees of hypothyroidism, which contrasts with the 75% decrease observed in the TRβ2 KO mice. Thus, the impact of the absence of the receptor on ligand-independent activation in vivo is greater than that observed in the presence of a powerful dominant-negative mutant TR. These 2 independent lines of evidence indicate that ligand-independent activation of the TSH-subunit genes can be modulated in vivo by altering TR expression and function, and they provide compelling evidence that TRs mediate ligand-independent activation of the TSHβ gene in vivo.
Thyroid hormone has long been known to be a potent inducer of GH gene expression (29
). TRβ2 is expressed in the somatotroph (28
), and there is evidence from studies in pituitary cell lines that TRβ2 is the major isoform mediating the stimulatory effect of thyroid hormone on GH gene expression (40
). Furthermore, in the absence of thyroid hormone, TRβ2 exhibits potent ligand-independent activation of GH expression — in contrast to TRβ1, which mediates ligand-independent repression (15
). The TRβ2 KO mice therefore provided a unique model with which to evaluate the role of TRβ2 in the regulation of GH gene expression by thyroid hormone in vivo. Interestingly, TRβ2 KO mice exhibited decreased basal GH expression and a blunted response to T3
. The small decrease in basal GH expression is very similar to the decrease in GH expression recently reported in mice lacking both TRβ isoforms (36
). This raises the possibility that the changes in GH expression in TRβ-null mice can be accounted for by loss of the TRβ2 isoform. It is important to note, however, that mice lacking both the TRα and TRβ isoforms exhibit profound downregulation of GH expression (36
), indicating that TRα and TRβ isoforms are both important in GH regulation by thyroid hormone.
Thyroid hormone plays an important role in auditory function. TRα and TRβ are expressed in the developing ear; however, the expression of TRβ is restricted to the cochlea (10
). Congenital hypothyroidism in humans is associated in many cases with deafness. In rodent models of congenital hypothyroidism, this is associated with structural abnormalities of the cochlea (41
). Genetic syndromes associated with loss of TRβ also result in deafness, which was a striking characteristic of the initial patient described with resistance to thyroid hormone; this patient had a homozygous deletion of TRβ (44
). More recently, Forrest et al. (21
) have demonstrated that genetic deletion of the entire TRβ locus in mice results in variable amounts of hearing loss. On average, ABR thresholds in TRβ-null mice were elevated by 20–40 dB. In individual animals, however, the threshold elevations ranged from a few decibels to complete loss of auditory responsiveness (25
). There were no gross structural cochlear abnormalities seen, implying that TRβ plays an important role in the maturation and maintenance of normal cochlear function but not in morphogenesis. Recent evidence suggests that the basis for the impaired hearing in TRβ-null mice is a developmental delay in the expression of the fast-activating potassium conductance (IK,f
) in inner hair cells (45
). Bradley et al. (10
) demonstrated coexpression of TRβ1 and TRβ2 in the developing cochlea in rats. Our mice, with selective deletion of the TRβ2 isoform of the TR, reveal that TRβ1 is sufficient to mediate normal cochlear development and function. These observations highlight important isoform-specific differences in the regulation of hearing by TRβ. Therefore, it will be important to determine whether the developmental regulation of IK,f
is normal in TRβ2-null mice.
The lack of an impact of TRβ2 deletion on hearing stands in contrast to its important role in the pituitary. The precise molecular mechanisms by which TRs regulate the development of hearing are unknown. As the target genes for the TR are identified in the cochlea, important insights into the factors governing the isoform-specific differences in gene regulation are likely to be obtained. A potential reason for the differential effects may lie in differences in the relative expression and relative roles of accessory cofactors in the ear versus the pituitary. It is known that retinoid receptors are expressed within the developing ear and are important heterodimer partners with the TRs on positively regulated genes (46
). We have previously shown that TRβ1 and TRβ2 exhibit distinct functional interaction with RXRs and N-CoR (19
). On negative TREs, the transcriptional activity of TRβ1 can be modified by liganded RXR, whereas that of TRβ2 cannot (19
). We have also shown that ligand-independent activation by TRβ1 and TRα1 is masked by corepressors, whereas the ligand-independent activation by TRβ2 is not (20
). Hence, tissue-specific differences in the expression and distribution of accessory cofactors may partly explain the differential roles of these TR isoforms in tissues in which they are coexpressed.
In summary, these studies have demonstrated that in keeping with its restricted expression, TRβ2 plays a central role in the regulation of the hypothalamic-pituitary-thyroid axis, and some role in the regulation of GH. In the thyrotroph, the function of TRβ2 cannot be replaced by the other TR isoforms. In the auditory system, however, normal hearing develops in the absence of TRβ2.