Dominant Inhibition of Thyroid Hormone Action Selectively in the Pituitary of Thyroid Hormone Receptor-ß Null Mice Abolishes the Regulation of Thyrotropin by Thyroid Hormone

E. Dale Abel, Egberto G. Moura, Rexford S. Ahima, Angel Campos-Barros, Carmen C. Pazos-Moura, Mary-Ellen Boers, Helen C. Kaulbach, Douglas Forrest and Fredric E. Wondisford

Division of Endocrinology (E.D.A.), Metabolism and Diabetes and Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah 84112; Departamento de Ciencias Fisiologicas (E.G.M.), Instituto de Biologia, Universidade do Estado do Rio de Janeiro, 20550-030 Rio de Janeiro, Brazil; Division of Endocrinology (R.S.A.), Diabetes and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; Department of Human Genetics (A.C.-B., D.F.), Mount Sinai School of Medicine, New York, New York 10029; Laboratorio de Endocrinologia Molecular (C.C.P.-M.), Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21000-000 Rio de Janeiro, Brazil; Division of Endocrinology and Metabolism (M.-E.B., H.C.K.), Beth Israel Deaconess Medical Center, Boston Massachusetts 02215; and Section of Endocrinology and Metabolism (F.E.W.), Pritzker School of Medicine, The University of Chicago, Chicago, Illinois 60637

Address all correspondence to: E. Dale Abel, Division of Endocrinology, Metabolism and Diabetes and Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, 15 North 2030 East, Building 533, Room 3410B, Salt Lake City, Utah 84112. E-mail: dale.abel{at}hmbg.utah.edu. Address reprint requests to: Fredric E. Wondisford, Section of Endocrinology and Metabolism, Pritzker School of Medicine, The University of Chicago, 5841 South Maryland Avenue MC1027, Chicago, Illinois 60637.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormones, T4 and T3, regulate their own production by feedback inhibition of TSH and TRH synthesis in the pituitary and hypothalamus when T3 binds to thyroid hormone receptors (TRs) that interact with the promoters of the genes for the TSH subunit and TRH. All TR isoforms are believed to be involved in the regulation of this endocrine axis, as evidenced by the massive dysregulation of TSH production in mice lacking all TR isoforms. However, the relative contributions of TR isoforms in the pituitary vs. the hypothalamus remain to be completely elucidated. Thus, to determine the relative contribution of pituitary expression of TR-{alpha} in the regulation of the hypothalamic-pituitary-thyroid axis, we selectively impaired TR-{alpha} function in TR-ß null mice (TR-ß-/-) by pituitary restricted expression of a dominant negative TR-ß transgene harboring a {Delta}337T mutation. These animals exhibited 10-fold and 32-fold increase in T4 and TSH concentrations, respectively. Moreover, the negative regulation of TSH by exogenous T3 was completely absent and a paradoxical increase in TSH concentrations and TSH-ß mRNA was observed. In contrast, prepro-TRH expression levels in T3-treated TR-ß-/- were similar to levels observed in the {Delta}337/TR-ß-/- mice, and ligand-independent activation of TSH in hypothyroid mice was equivalently impaired. Thus, isolated TR-ß deficiency in TRH paraventricular hypothalamic nucleus neurons and impaired function of all TRs in the pituitary recapitulate the baseline hormonal disturbances that characterize mice with complete absence of all TRs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE REGULATION OF ambient thyroid hormone concentrations depends upon the orchestrated interactions between thyroid hormone (T3), hypothalamic TRH, and TSH (1). The synthesis of TRH and TSH are negatively regulated by thyroid hormone that acts by binding to thyroid hormone receptors (TRs), which interact with the promoters of the TRH and TSH subunit genes (2, 3). Three T3-binding isoforms of the TR, TR-{alpha}1, TR-ß1, and TR-ß2, are expressed within the thyrotroph and the TRH neurons of the paraventricular hypothalamic nucleus (PVN) (4, 5, 6). It is unclear, however, if these isoforms play equivalent roles in the regulation of TRH and TSH production in vivo.

Recent studies in transgenic and knockout (KO) mice have provided some insight into the relative roles of TR isoforms in the regulation of TSH and have suggested important differences in the roles of the pituitary vs. the hypothalamic TRH neurons in regulating ambient thyroid hormone concentrations. We demonstrated that selective expression of a mutant thyroid hormone receptor in the pituitaries of transgenic mice resulted in increased production of TSH, which was only partially suppressible by T3 (7). Paradoxically, ambient thyroid hormone concentrations were only marginally elevated and TRH expression was down-regulated. Thyroid hormone levels became elevated only after the exogenous administration of TRH. These data underscored the important role of the pituitary in regulating TSH concentrations, and the essential role of TRH expression in modulating TSH bioactivity. Targeted ablation of the TR-ß isoform (TR-ß-/-), resulted in elevated TSH concentrations, which were partially suppressible by T3 treatment (8, 9).

The degree of insensitivity of TSH to T3 suppression in TR-ß-/- mice is similar in magnitude to that observed in our mice with pituitary expression of a mutant TR (7). In contrast to the pituitary mutant mice, basal thyroid hormone concentrations were 2- to 3-fold higher in TR-ß-/- mice, suggesting that absence of the TR-ß isoform in the TRH neuron results in dysregulated TRH production that ultimately drives the hyperthyroxinemia.

Similar changes were also observed after targeted ablation of the TR-ß2 (TR-ß2-/-) (10); the TR-ß2 isoform is most highly expressed in the pituitary and the hypothalamus (11). Analysis of TRH gene expression in TRH-producing neurons of the PVN of TR-ß2-/- mice confirmed dysregulation of TRH expression and lack of responsiveness to T3 (12). The partial responsiveness of TSH to exogenous T3 in the three mouse models described above, particularly the observations in mice that lack all TR-ß isoforms, suggests that the residual TR-{alpha}1 isoform in thyrotrophs, retains an important role in the negative regulation of TSH by T3 (13). Additional evidence for a permissive role of TR-{alpha}1in regulating the pituitary-thyroid axis comes from the observation that increasing the expression of TR-{alpha}1 in TR-ß null mice normalized abnormalities in T4, T3, and TSH concentrations (14).

Mice with targeted ablation of TR-{alpha}1 or of the entire TR-{alpha} locus develop only minor changes in thyroid hormone and TSH concentrations, and thyroid hormone and TSH levels appear to be lower than normal (15, 16). Mice that lack all TR-{alpha} isoforms exhibit increased sensitivity of the thyrotroph to T3, which is believed to be due to loss of the constitutive silencing by TR-{alpha}2 (17). The lack of dramatic changes in thyroid hormone levels in the absence of TR-{alpha} initially raised the possibility that this isoform did not play an important role in the regulation of the hypothalamic-pituitary-thyroid axis. However, the generation of mice that lack all TR-ß and TR-{alpha} isoforms suggests a regulatory role for TR-{alpha} in that compound TR-ß/{alpha} KO mice develop massive elevations of TSH in the range observed in hypothyroidism and equally dramatic elevations in thyroid hormone concentrations (18, 19, 20). The development of these striking abnormalities strongly point to a previously unrecognized but important role of the TR-{alpha} isoform in the regulation of the hypothalamic-pituitary-thyroid axis. It is unknown if the defective regulation of the thyroid axis in these mice is due primarily to the absence of the TR-{alpha} and ß isoforms in the pituitary or the hypothalamus or whether this represents impairment in both organs.

Thus, to understand the relative roles of the pituitary vs. the hypothalamus in the regulation of thyroid function, a mutant TR transgene associated with generalized resistance to thyroid hormone (RTH) was selectively targeted to the pituitaries of TR-ß-/- mice. These mice lack TR-ß isoforms in the hypothalamus and pituitary gland, but have intact TR-{alpha} expression. The presence of the mutant TR transgene in the pituitary would be expected to dominantly inhibit the function of the residual TR-{alpha} selectively in the pituitary but not in the PVN, thereby providing a unique opportunity to carefully define the role of pituitary TR expression in the regulation of the thyroid axis. Herein we report that targeted inhibition of pituitary thyroid hormone receptors in TR-ß-/- mice results in complete dysregulation of TSH synthesis leading to massive hyperthyroxinemia of similar magnitude to that reported in mice with no functional TRs. These data underscore the complex interactions between anatomical location and regional differences in isoform-specific responses of receptors that participate in endocrine feedback loops.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Basal Thyroid Hormone Concentrations
As previously reported, T4 concentrations were not significantly increased in heterozygous TR-ß null mice (TR-ß+/-) relative to wild-type (WT) controls (4.0 ± 0.3 vs. 3.5 ± 0.4 µg/dl) and were modestly increased (5.7 ± 0.3 µg/dl) in {Delta}337T transgenic mice ({Delta}337/TR-ß+/+) (Fig. 1Go). Haploinsufficiency at the TR-ß locus in combination with the pituitary expression of the {Delta}337T transgene ({Delta}337/TR-ß+/-) increased basal T4 concentrations to the same extent as was observed in TR-ß-/- (10.2 ± 0.6 and 11.3 ± 0.9 µg/dl, respectively). Expression of the {Delta}337T transgene in the pituitaries of TR-ß-/- mice ({Delta}337/TR-ß-/-) resulted in massively elevated T4 concentrations of 33.9 ± 1.9 µg/dl. Total T3 concentrations followed a similar pattern, with 2.3- to 2.5-fold increases in TR-ß-/- and {Delta}337/TR-ß+/- mice, respectively, whereas T3 concentrations in {Delta}337/TR-ß-/- were increased 7-fold relative to WT controls (Fig. 1Go). TSH concentrations were 1.9-, 2.3-, and 3.4-fold higher than WT controls in TR-ß-/-, {Delta}337/TR-ß+/+ and {Delta}337/TR-ß+/-, respectively. TSH concentrations in {Delta}337/TR-ß+/- were significantly higher than values obtained in TR-ß-/- mice. However, TSH concentrations were dramatically increased by 32-fold in {Delta}337/TR-ß-/- relative to WT controls (1945 ± 249 ng/ml vs. 61 ± 9 ng/ml) (Fig. 1Go).



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Fig. 1. Basal T4, T3, and TSH Concentrations in Mice with Various Genotypes as Shown

In this and all other figures, +/+ means WT for the TR-ß locus (TR-ß+/+), +/- means heterozygous for the TR-ß locus (TR-ß+/-), -/- means TR-ß null (TR-ß-/-) and {Delta} indicates the presence of the {Delta}337T transgene. {dagger}, P < 0.0001 vs. all other genotypes; ¶, P < 0.01 vs. +/+ and +/- (T4); *, P < 0.02 vs. +/+; +/- and {Delta}+/+ (T4 and T3); §, P < 0.02 vs. -/-; and {ddagger}, P < 0.01 vs. +/+ and +/- (TSH). Numbers of animals in each group are as follows. T4, +/+ = 11; {Delta}+/+ = 12, +/- =13, {Delta}+/- = 19, -/- = 18, {Delta}-/- = 14. T3, +/+ = 4, +/- =10, {Delta}+/- = 7, -/- = 10, {Delta}-/- = 6. TSH, +/+ = 11, {Delta}+/+ = 12, +/- =12, {Delta}+/- = 15, -/- = 11, {Delta}-/- = 13.

 
Analysis of in Vivo Regulation of Thyrotroph Function by T3
Administration of T3 suppresses endogenous TSH secretion and ultimately T4 production by the thyroid gland. We therefore sought to determine the sensitivity of the hypothalamic-pituitary-thyroid axis in transgenic mice by measuring total T4 concentrations at weekly intervals during 3 wk of ip administration of pharmacological doses of T3. TSH concentrations were also measured at the end of the period of T3 treatment. In WT and TR-ß+/- mice, exogenous T3 completely suppressed both T4 and TSH concentrations. As previously reported (7), partial suppression of T4 and TSH was observed in {Delta}337/TR-ß+/+ mice. {Delta}337/TR-ß+/- and TR-ß-/- mice had a greater degree of central RTH than the preceding genotypes (Fig. 2Go, A and C).



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Fig. 2. Effect of T3 Administration on Serum T4 and TSH Concentrations

A, Total T4 concentrations obtained at weekly intervals during administration of pharmacological doses of T3 in genotypes as indicated. {Delta} +/+ curve is significantly higher than +/+ and +/- curves at 2- and 3-wk time points (P < 0.003). {Delta}+/- and -/- curves are significantly higher than {Delta}+/+, +/+ and +/- curves at 2- and 3-wk time points (P < 0.001). {dagger}, P < 0.0001 vs. respective genotype at 0 wk for all curves. B, Same data as panel A with the addition of data from {Delta}-/- mice. {Delta}-/- curve is significantly higher at all time points, than all other curves (P < 0.0001). *, P < 0.0004 vs. {Delta}-/- at 0 wk. C, TSH concentrations obtained before the initial dose of T3 and at the end of 3 wk of T3 administration in the genotypes as indicated. {ddagger}, P < 0.06; ¶, P < 0.0001 vs. T3-treated of the same genotype. Numbers of animals in each group are as follows. T4 basal, +/+ = 11, {Delta}+/+ = 12, +/- = 13, {Delta}+/- = 19, -/- = 18, {Delta}-/- = 14. T4, 1 wk: +/+ = 11, {Delta}+/+ = 12, +/- = 8, {Delta}+/- = 7, -/- = 18, {Delta}-/- = 9. T4 2-wk, +/+ = 11, {Delta}+/+ = 10, +/- = 8, {Delta}+/- = 7, -/- = 18, {Delta}-/- = 9. T4 3-wk: +/+ = 11, {Delta}+/+ = 12, +/- =8, {Delta}+/- = 7, -/- = 18, {Delta}-/- = 9. TSH: +/+ = 11, {Delta}+/+ = 12, +/- =4, {Delta}+/- = 4, -/- = 4, {Delta}-/- = 4.

 
In contrast, {Delta}337/TR-ß-/- exhibited a paradoxical increase in both total T4 and TSH response indicating that negative regulation of the hypothalamic-pituitary-thyroid axis was completely abolished and potentially reversed (Fig. 2Go, B and C). Total T3 concentrations were also increased in {Delta}337/TR-ß-/- mice 8 h after the last T3 injection, rising from 749 ± 70 at baseline to 1091 ± 126 ng/dl (P < 0.05). This contrasts with a tendency for T3 levels to decline after treatment with T3 in all other genotypes (data not shown). TSH-ß mRNA was markedly induced in pituitaries from {Delta}337/TR-ß-/- mice and did not decrease after T3 administration. This contrasts with partial suppression in {Delta}337/TR-ß+/+ mice and complete suppression of TSH-ß expression in WT mice, which is consistent with our earlier observations (7) (Fig. 3Go).



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Fig. 3. TSH-ß Response to T3 Administration

A, Representative Northern blots of pooled pituitary RNA, showing expression of TSH-ß mRNA before and after T3 administration in the genotypes as shown. Fifteen micrograms of RNA were loaded in each lane. There were no differences in loading and representative cyclophilin expression is shown. B, Densitometric analyses of Northern blots were performed and corrected for cyclophilin expression. Data are expressed as arbitrary OD units.

 
Analysis of the in Vivo Regulation of Prepro-TRH Expression by T3
Given the elevated T4 and T3 concentrations in {Delta}337/TR-ß-/- mice, it was important to determine prepro-TRH gene expression in all mice under ambient conditions and after 3 wk of T3 treatment (Fig. 4Go, A–C). Under basal conditions, total prepro-TRH expression was significantly decreased by approximately 15% in {Delta}337/TR-ß+/+ relative to TR-ß+/+, which supports our previously published results (7). More importantly, basal TRH expression was significantly increased in TR-ß-/- and {Delta}337/TR-ß-/- relative to both TR-ß+/+ and {Delta}337/TR-ß+/+ and did not change after T3 treatment. This contrasts with T3 mediated suppression of prepro-TRH expression in TR-ß+/+ and {Delta}337/TR-ß+/+ mice. Thus, prepro-TRH expression does not respond to T3 administration in the absence of TR-ß, which supports our previously published data that TRH response across a range of thyroid hormone levels from hypothyroidism to T3suppression is absent in mice that lack TR-ß2 (12). Thus, under conditions of hyperthyroxinemia prepro-TRH expression is significantly elevated in mice that lack TR-ß irrespective of the presence or absence of the of {Delta}337/TR transgene. We also observed regional differences in prepro-TRH expression with greater T3 suppression observed in the caudal than rostral PVN in mice with the WT TR-ß allele, relative to those that were null for TR-ß (Fig. 4Go, A and B).



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Fig. 4. Prepro-TRH Expression

A, Representative dark-field photomicrographs showing prepro-TRH mRNA in the rostral and caudal PVN of WT mice (TR-ß+/+) (a and c), and TR-ß-/- mice (b and d), after 3 wk of T3 administration. Scale bar, 300 µm. B, Representative dark-field photomicrographs showing prepro-TRH mRNA in the rostral and caudal PVN of {Delta}337T transgenic mice on the WT TR-ß background ({Delta}TR-ß+/+) (a and c), and {Delta}337T transgenic mice on the TR-ß-/- background ({Delta}TR-ß-/-) (b and d), after 3 wk of T3 administration. Scale bar, 300 µm. C, Relative total (caudal + rostral) pre-pro TRH expression as assessed by laser densitometry in the PVN under ambient conditions (Basal) and after T3 treatment in mice of the genotypes shown. n = 4 mice per group. {dagger}, P < 0.0001 vs. basal of same genotype; *, P < 0.01 vs. +/+ basal; **, P < 0.0001 vs. +/+ and {Delta}+/+ basal; {ddagger}, P < 0.0001 vs. +/+ and {Delta}+/+ T3 treated).

 
Analysis of in Vivo Regulation of Thyrotroph Function in Hypothyroid Mice
Three weeks after radioactive iodine ablation, all animals had T4 levels that were at or below the limit of detection of the T4 assay (data not shown). TSH concentrations were increased more than 300-fold in WT mice (P < 0.001 vs. all other genotypes) (Fig. 5Go). In contrast, the increase in TSH concentrations was markedly blunted in all other mice. TSH concentrations increased by 100-fold in TR-ß+/-, 53-fold in TR-ß-/-, 33-fold in {Delta}337/TR-ß+/+, 30-fold in {Delta}337/TR-ß+/- but by only 2.2-fold in {Delta}337/TR-ß-/-. Differences in the fold increase are largely accounted for by differences in the basal concentration of TSH, as all mutant mice achieved similar absolute levels of TSH in response to hypothyroidism. Thus, the 2.2-fold increase in the {Delta}337/TR-ß-/- can be explained on the basis of high basal TSH levels observed in these animals. In summary, dominant negative inhibition of pituitary TRs, haploinsufficiency or loss of TR-ß all lead to impaired ligand-independent activation of TSH production in vivo.



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Fig. 5. Serum TSH Response to Hypothyroidism

TSH concentrations were measured before (basal, open bars) and 4 wk after mice were rendered hypothyroid by I131 administration (hypothyroid, closed bar). {dagger}, P < 0.0001 vs. all other genotypes (hypothyroid TSH). Numbers of animals in each group are as follows. TSH: +/+ = 11, {Delta}+/+ = 11, +/- =7, {Delta}+/- = 6, -/- = 11, {Delta}-/- = 5.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
By expressing the {Delta}337T mutant TR selectively in the pituitaries of TR-ß-/- mice, we demonstrate important differential roles for TR isoform expression in the pituitary vs. the hypothalamus in the regulation of the hypothalamic-pituitary thyroid axis by T3. Expression of the mutant transgene selectively in the pituitary of TR-ß-/- mice results in dominant negative inhibition of the residual TR-{alpha} isoforms in the pituitary. We have previously shown by RT-PCR that expression of the mutant TR transgene is limited to the pituitary and is not present in the hypothalamus (7). We therefore believe that the function of TR-{alpha} is preserved in PVN neurons. The impact of this manipulation on the thyroid hormone axis is a marked increase in T4, T3, and TSH concentrations that are very similar to those observed in mice that lack both TR-ß and TR-{alpha} isoforms or mice that are homozygous for germ line expression of the {Delta}337T mutation (21). Moreover, responsiveness of the thyrotroph to exogenous T3 is completely abolished and ligand-independent activation of the TSH gene by hypothyroidism in vivo is markedly impaired. These studies underscore the complex interplay between isoform specific receptor activity and receptor location in the regulation of the hypothalamic-pituitary-thyroid axis. Furthermore, our findings highlight important region-specific and isoform-specific responses to ligand-dependent as well as ligand-independent receptor signaling. This unique model has allowed us to directly dissect these mechanisms in a way that has not been possible to date in various TR mutant mice (germ line null or germ line expression of dominant negative mutants).

Mice that lack TR-{alpha} and TR-ß (TR-{alpha}1-/--/- or TR-{alpha}o/o-/-), exhibit profound dysregulation of the hypothalamic-pituitary-thyroid axis (18, 19, 20). Because all TR isoforms are lacking in both the pituitary and PVN neurons, it is impossible to know if the hormonal dysregulation reflects impairment in both the pituitary gland and the PVN. Mice that are heterozygous for a germ line {Delta}337T mutant TR retain a single copy of the WT TR-ß allele and all TR-{alpha} gene products in both the pituitary and the PVN. Thyroid hormone abnormalities in these mice are modest and similar to that observed in TR-ß-/- mice (21). These mice therefore do not recapitulate the hormonal abnormalities observed in mice that lack all TR isoforms. Thus, heterozygous germ line expression of {Delta}337T only partially neutralizes the function of the residual TR-{alpha} and the residual TR-ß alleles by mechanisms that may involve constitutive interactions with accessory cofactors such as nuclear corepressors (21).

In contrast, homozygous germ line {Delta}337T mutant mice develop elevations in T4, T3, and TSH that are similar to those observed in mice that lack all TRs (21), indicating that homozygous expression of this mutant TR effectively inhibits the function of TR-{alpha}. However, in this model it is also impossible to discern if dysregulated function in the pituitary and PVN contribute equally to the hormonal phenotype observed. By expressing the {Delta}337T mutant selectively in the pituitaries of TR-ß-/- mice, our results suggest that inhibition of TR-{alpha} expression in the pituitary is the basis for the complete dysregulation of thyrotroph responsiveness to T3. These data are also consistent with the conclusion that that loss of expression of TR-{alpha} in the PVN may contribute little or not at all to the hormonal abnormalities that characterizes mice that lack all TR isoforms or are homozygous for a germ line {Delta}337T mutation. The possibility also exists, however, that the pituitary effect may be dominant, and this may render it difficult to discern any effect of TR-{alpha} expression on the regulation of TRH, unless this is studied in isolation. Expressing the {Delta}337T selectively in the PVN of TR-ß null mice using the mouse TRH promoter could allow for such an analysis. The findings in TR-ß-/- mice in this present study and in our previous study of TR-ß2 KO mice (12) in which we observed no effect of T3 to repress TRH gene expression, argues, however, in favor of a limited role of TR-{alpha} expression in the regulation of TRH gene expression by T3.

The responsiveness of the thyrotroph to T3 administration in vivo was assessed by administering T3 exogenously for 3 wk. WT and TR-ß+/- mice exhibited complete suppression of serum T4 and TSH under these conditions, further confirming that one allele of the TR-ß locus is sufficient to impart normal responsiveness of the hypothalamic-pituitary-thyroid axis to thyroid hormone. Expression of the {Delta}337T transgene in the pituitary of WT mice modestly impairs thyrotroph responsiveness to T3, but to a lesser degree than that observed in the TR-ß-/- mice, whereas expression of the {Delta}337T transgene in TR-ß+/- mice recapitulates the pituitary resistance that is seen in TR-ß-/- mice. Taken together, these observations suggest that the mutant TR transgene is able to inhibit more than 50% of TR-ß function, and in the absence of TR-ß also inhibits TR-{alpha} function. This conclusion is further supported by the fact that there is no suppression of serum T4 or TSH when the {Delta}337T transgene is expressed in TR-ß-/- mice.

Not only was negative regulation of T4 and TSH by T3 completely lacking, but we also observed a paradoxical increase in serum TSH, T4, and TSH-ß mRNA after T3 administration. These findings indicate that, in the regulation of the hypothalamic-pituitary thyroid axis all T3-mediated feedback may not necessarily be negative. The mechanisms for these unexpected observations are not immediately apparent. Possible explanations include: 1) A nongenomic effect of T3 on thyrotroph cells that ultimately enhance TSH gene expression (increased transcription or enhanced message stability) that only becomes manifest when nuclear receptors are inactivated in these cells. 2) An indirect effect of T3 activation in sites that are remote from the pituitary that in turn results in activation of TSH gene expression. These effects could be paracrine, endocrine or neural. 3) An as-yet-undescribed nuclear TR isoform that positively regulates the TSH promoter. The design of the present study does not allow us to differentiate between these possibilities. Our novel observations should provide the basis for analyses of TSH and T4 responses to exogenous T3 administration in mice that are devoid of all known TRs. The lack of a similar response in those mice would support the hypothesis of a remote T3-mediated effect that could account for the positive regulation of TSH and T4, by T3 in our mice.

Because T4 levels were persistently elevated in {Delta}337/TR-ß-/- mice, prepro-TRH expression was determined in all mice after 3 wk of T3 treatment. As expected, total PVN TRH expression was significantly reduced in T3-treated WT mice. We observed regional differences in the prepro-TRH gene expression with greater T3-mediated suppression observed in the caudal PVN of mice that are WT for the TR-ß allele relative to those that are null for the TR-ß allele; with suppression being greatest in mice that harbored the {Delta}33T transgene ({Delta}337/TR-ß+/+). These changes are consistent with our earlier observations in which we observed that TRH expression was also reduced in mice with pituitary expression of the {Delta}337T transgene under basal conditions, and more markedly in the caudal than the rostral PVN (7). Differences in basal suppression of prepro-TRH expression between {Delta}337/TR-ß+/+ mice and controls in the current study are less than that observed by us previously (7). This may reflect biological variability or technical differences in the in situ hybridization techniques between studies. We are reassured however by the observation that the reduction in prepro-TRH expression in T3-treated {Delta}337/TR-ß+/+ of 33%, relative to control mice is similar in magnitude to our earlier report. Prepro-TRH expression levels were similarly elevated in TR-ß-/- and {Delta}337/TR-ß-/- mice relative to WT controls and were not altered by T3 administration. These observations indicate that TR-ß isoforms are required for mediating the suppression of TRH gene expression by T3. These data are also consistent with and support our earlier observations that TR-ß2 (the expression of which is absent in TR-ß-/- mice) is the major mediator of the negative regulation of TRH expression by thyroid hormone (12).

Our studies also confirm an important role for increased TRH tone in mediating the hormonal disturbances that characterizes RTH. We have previously demonstrated that expression of the {Delta}337T transgene in the pituitaries of mice that were WT at both TR-ß and TR-{alpha} loci, was associated with insignificant or marginal changes in thyroid hormone concentrations despite a 2-fold elevation in TSH concentrations (7). Moreover, T4 concentrations were increased only after the administration of exogenous TRH. These observations suggested that reduced TR-ß mediated signaling in the PVN might increase TRH expression and release sufficiently to enhance the bioactivity of TSH, presumably by changing its glycosylation pattern. Haploinsufficiency of the TR-ß locus (TR-ß+/-) does not lead to any significant changes in T4, T3, or TSH concentrations. However, pituitary expression of the {Delta}337T transgene in TR-ß+/- mice leads to significant elevations in T4, and T3 despite the fact that TSH concentrations were not significantly different from those of {Delta}337/TR-ß+/+ mice. Moreover, increased basal prepro-TRH expression in TR-ß-/- mice and higher T4 concentrations in TR-ß-/- mice than in {Delta}337/TR-ß+/+ mice despite similar concentrations of TSH provides further compelling evidence for the role that increased TRH tone plays in raising T4 concentrations in RTH. The fact that T4, T3, and TSH concentrations were not increased by expression of the pituitary {Delta}337T transgene in TR-ß+/- mice to the same extent as that observed when expressed in TR-ß-/- mice suggests that the presence of the residual WT TR-ß allele in {Delta}337/TR-ß+/- mice is sufficient to impart responsiveness to the thyrotroph.

Another important finding of our study is the observation that ligand-independent activation of TSH expression can be impaired in vivo. Moreover, our study indicates that ligand-independent activation of TSH subunit gene expression is more sensitive to changes in TR-ß expression or function than is T3 mediated ligand-dependent suppression. Thus, whereas ligand-dependent suppression of serum TSH levels is normal in TR-ß+/- mice, ligand-independent activation of TSH production by hypothyroidism is markedly impaired. Indeed, haploinsufficiency for the TR-ß locus impairs ligand-independent activation of TSH production in vivo to the same extent as total absence of TR-ß in vivo. In contrast to the effect on ligand-dependent repression, the presence or absence of the {Delta}337T pituitary transgene does not lead to any significant additional impairment in vivo of ligand-independent activation in TR-ß+/- or TR-ß-/- mice. Thus, the dominant negative transgene exhibits differential effects on ligand-dependent repression vs. ligand-independent activation of the TSH subunit genes. The molecular mechanisms that govern ligand-independent activation of the promoter of the TSH subunit genes are partially understood. Our novel in vivo observations would indicate that the stoichiometry of TR-ß isoforms and the cofactors with which they interact to mediate ligand-independent activation of TSH subunit genes are tightly regulated in vivo.

In summary, these studies indicate that normal function of all pituitary TR isoforms is essential for the regulation of thyrotroph responsiveness to thyroid hormone. Our data also suggest that any role that TR-{alpha} plays in the regulation of TRH expression by thyroid hormone might be minimal or negligible. Thus, the dramatic dysregulation of the hypothalamic-pituitary-thyroid axis that exists in mice that lack all TRs or in those that are homozygous for germ line mutations of TR-ß reflect the impact of total TR deficiency or dysfunction in the pituitary acting in concert with TR-ß dysfunction/deficiency in the paraventricular TRH neurons (Table 1Go). Our findings underscore the complex interplay between anatomical location as well as regional and isoform specific differences in receptor signaling in the feedback regulation of the hypothalamic-pituitary-thyroid axis.


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Table 1. Summary of Transgenic and Knockout Mouse Models with Altered TR-Isoform Expression or Function in the Hypothalamus and Pituitary

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
The Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center (Boston, MA) approved all aspects of animal care and experimentation performed in this study. Animals were maintained on a 12-h light/12-h dark schedule (light on at 0600) and fed laboratory chow and water ad libitum. Blood was obtained for total T4, total T3, and TSH from the tail vein. Experimental hypothyroidism was induced by administering 150 µCi of 131I by ip injection to mice who had been placed on a low iodine diet (Harlan Teklad, Madison, WI) for 8 d. Suppression of pituitary TSH and hypothalamic TRH production was attempted by daily ip injections of T3 (1 µg/ml) in buffered HEPES for 3 wk at doses of 0.2 µg/100 g mouse body weight during the wk 1, 0.5 µg/100 g mouse body weight during wk 2, and 1.0 µg/100 g mouse body weight during wk 3.

Generation of Transgenic Mice
Transgenic mice (WT for the TR-ß allele) in which the mouse {alpha}-subunit promoter was used to express the naturally occurring human mutant TR {Delta} 337T ({Delta}337/TR-ß+/+) selectively and exclusively in the pituitary (7) were crossed with TR-ß null mice (TR-ß-/-) (8). We have previously shown by RT-PCR that expression of the mutant TR transgene is limited to the pituitary and is not present in the hypothalamus (7). All offspring of the initial crosses were heterozygous for the TR-ß null allele and some also harbored the transgene. Heterozygous transgenics ({Delta}337/TR-ß+/-) were then crossed with heterozygous nontransgenics (TR-ß+/-). This approach yielded offspring with the following six genotypes: {Delta}337/TR-ß+/+, {Delta}337/TR-ß+/-, {Delta}337/TR-ß-/-, TR-ß+/+, TR-ß+/-, and TR-ß-/-. Given the average size of mouse litters, it would be impossible for a single litter to provide sufficient numbers of mice from of all genotypes for meaningful analysis. Thus we set up multiple breeding cages of {Delta}337/TR-ß+/- mice with TR-ß+/- mice, and studied all of the offspring. Therefore, littermates (across multiple litters) were used as controls. Because the parental mice were all derived from the offspring of initial crosses of TR-ß-/- and {Delta}337/TR-ß+/+ mice, we believe that this paradigm minimizes the likelihood that genetic heterogeneity confounded the analysis of our data. Because we studied all mice that were generated, the numbers in each group will not necessarily be equal. Mice were genotyped by Southern blotting for the presence of the transgene and for the presence of the TR-ß WT and null alleles as previously described (7, 8).

RNA Analysis
RNA was extracted from pooled pituitaries (five to eight animals), using guanidium thiocyanate and selective precipitation and isopycnic centrifugation with lithium chloride and cesium trifluoroacetate (Pharmacia Biotech, Piscataway, NJ). Pituitaries were obtained before and after treatment of mice with T3. Fifteen micrograms of RNA were resolved on a 1.2% formaldehyde agarose gel and transferred to a nylon membrane. The membrane was hybridized to a radiolabeled mouse TSH-ß subunit cDNA probed under high stringency conditions.

Localization and Quantification of Prepro-TRH mRNA by in Situ Hybridization Histochemistry
After treatment with exogenous T3, mice of the following genotypes ({Delta}337/TR-ß+/+, {Delta}337/TR-ß-/-, TR-ß+/+, and TR-ß-/-) (four per group) were anesthetized with an ip injection of sodium pentobarbital (50 mg/kg). Sham-treated mice of the same genotypes were used as controls. They were perfused transcardially with PBS prepared with diethylpyrocarbonate-treated water, followed by 10% neutral buffered formalin. Brains were removed, immersed in the same fixative overnight and then cryoprotected in 20% sucrose in PBS-diethylpyrocarbonate at 4 C. Five series of 20 µm coronal sections were cut on a tabletop cryotome and mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA). The protocol for in situ hybridization histochemistry has been described previously (12). Prepro-TRH (prepro-TRH) mRNA was localized using a 35S-uridine triphosphate-labeled cRNA probe synthesized from a plasmid containing the cDNA for the mouse prepro-TRH gene (kind gift of Dr. Masatomo Mori (Gunma University, Maebashi, Japan). Control sections were hybridized with a sense cRNA probe.

The slides from various groups were processed for in situ hybridization under the same conditions. After posthybridization washing and air-drying, the slides were exposed to film (Biomax MR, Eastman Kodak Co., Rochester, NY), and subsequently developed under the same conditions. The signal corresponding to prepro-TRH mRNA levels through the rostrocaudal extent of the PVN was analyzed on film autoradiograms, corresponding to bregma levels -0.58 mm, -0.82 mm, and -1.22 mm, respectively (Figs. 36, 38, and 41 of Franklin and Paxino’s atlas) (22). Autoradiograms were scanned and analyzed blindly by computerized laser densitometry, using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA), n = 4 mice per group. At each level, a rectangle was drawn to enclose the PVN, and the absorbance of the autoradiographic signal was measured. The rectangle was then reproduced to the same dimension and used to measure absorbances in similar regions in matched sections from other animals. Mean absorbance values corresponding to the content of TRH mRNA in PVN were corrected for background (nonspecific signal) by subtracting the absorbance in the cerebral cortex on the same brain section not known to express TRH mRNA. For photomicrography, emulsion-dipped slides were examined with a Nikon E600 microscope, and dark-field photomicrographs were taken with a SPOT RT digital camera (Phase 3 Imaging Systems, Glen Mills, PA).

RIA
Total T4 levels were measured in 10-µl serum samples in duplicate determinations by a mouse specific RIA (ImmuChem-coated tube-T4125I RIA Kit, ICN Pharmaceuticals, Costa Mesa, CA.). Total T3 was measured in 100-µl serum samples by RIA (ImmuChem-coated tube-T3125I RIA Kit, ICN Pharmaceuticals). TSH was measured in 25 µl serum samples in triplicate determinations by a specific mouse TSH RIA using a mouse TSH/LH reference preparation (AFP51718mp), a mouse TSH antiserum (AFP98991), and rat TSH antigen for radioiodination (National Institute of Diabetes Digestive and Kidney Diseases, rTSH-I-9). All reagents were obtained from Dr. A. F. Parlow (Harbor University of California at Los Angeles Medical Center, Torrance, CA). The standard curve was performed in hyperthyroid mouse serum and the limit of sensitivity was less than 20 ng/ml. The inter- and intraassay variations were less than 6%.

Statistical Analysis
Data are expressed as means ± SEM. Differences between two groups were assessed by unpaired two-tail t tests and multiple comparisons were analyzed by ANOVA and significance assessed by Fisher’s protected least significant difference test.


    FOOTNOTES
 
This work was supported by NIH Grants DK-02458 (to E.D.A.), DC-03441 (to D.F.) and DK-49126 and 50564 (to F.E.W.). E.D.A. was the recipient of a Thyroid Research Advisory Council Award. E.G.M. and C.C.P.-M. were recipients of a CNPq grant from the government of Brazil. H.K. was supported by NIH Training Grant T32 DK-07561. This work was also supported, in part, by the March of Dimes Birth Defects Foundation (Grant No. 1-FY00-670) and a Hirschl Award (to D.F.). A.C.-B. was supported by Grant PF 97 0679951 of the Spanish Ministry of Education and Culture.

Current address for A.C.-B: Department of Pediatric Endocrinology, Hospital Infantil Universitario Niño Jesús (Madrid, Spain).

Abbreviations: KO, Knockout; PVN, paraventricular hypothalamic nucleus; RTH, resistance to thyroid hormone; TR, thyroid hormone receptor; WT, wild-type.

Received for publication March 28, 2003. Accepted for publication June 9, 2003.


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