Thyroid Hormone Receptor-Specific Interactions with Steroid Receptor Coactivator-1 in the Pituitary

Peter M. Sadow, Eugene Koo, Olivier Chassande, Karine Gauthier, Jacques Samarut, Jianming Xu, Bert W. O’Malley, Hisao Seo, Yoshiharu Murata and Roy E. Weiss

Departments of Medicine (P.M.S., E.K., R.E.W.) and Pathology (P.M.S.), The University of Chicago, Chicago, Illinois 60637; Laboratoire de Biologie Moleculaire et Cellulaire de l’Ecole Normale Supérieure de Lyon (O.C., K.G., J.S.), Lyon 69364, France; Department of Molecular and Cellular Biology (J.X., B.W.O’M.), Baylor College of Medicine, Houston, Texas 77030; and Research Institute of Environmental Medicine (H.S., Y.M.), Nagoya University, Nagoya 464-8601, Japan

Address all correspondence and requests for reprints to: Roy E. Weiss, M.D., Ph.D., Thyroid Study Unit, MC 3090, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois. E-mail: rweiss{at}medicine.bsd.uchicago.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid receptor coactivator-1 (SRC-1) is a transcription cofactor that enhances the hormone-dependent action mediated by the thyroid hormone (TH) receptor (TR) as well as other nuclear receptors. However, it is not known whether the SRC-1-mediated activation of TH-regulated gene transcription is TR isoform specific in the pituitary. We generated mice that were deficient in TR{alpha} and SRC-1 (TR{alpha}0/0SRC-1-/-), as well in TRß and SRC-1 (TRß-/-SRC-1-/-), and thyroid function tests and effects of TH deprivation and TH treatment were compared with wild-type mice or mice with deletion of either TRs or SRC-1 alone. We have shown that 1) TRß-/-SRC-1-/- mice demonstrate more severe TH resistance than either the SRC-1-/- or TRß-/- mice; the additive effect indicates that SRC-1 has an independent role in TH action over that of TRß; 2) SRC-1 facilitates TRß and TR{alpha}-mediated down-regulation of TSH, as TR{alpha}0/0SRC-1-/- mice demonstrate TH resistance rather than hypersensitivity as seen in TR{alpha}0/0mice; and 3) a compensatory increase in SRC-1 expression is associated with the TH hypersensitivity seen in TR{alpha}-deficient animals. We conclude that SRC-1 action in the pituitary mediates TH action via specific TR subtypes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THYROID HORMONE ACTION is mediated through intranuclear thyroid hormone receptors (TR) {alpha} and TRß. TRs function as transcription factors binding to thyroid hormone (TH) response elements (TREs) as monomers, homodimers, or heterodimers with other nuclear receptors such as the retinoid X receptors (1, 2, 3). Unliganded TR complexes associate with transcriptional corepressors that recruit histone deacetylases and facilitate tight, inaccessible DNA (4, 5). Upon binding to TH, a conformational change takes place within the TR complex, releasing the corepressor and allowing for binding of a coactivator. The coactivator has a number of functions that may include intrinsic histone acetyltransferase (HAT) activity, recruitment of HATs, additional transcription factors, and RNA polymerase (6, 7).

Several classes of nuclear coactivators have been described that are important in mediating the response of mammalian cells to steroid, thyroid, and retinoid hormones (8, 9, 10, 11, 12, 13). The p160 family of coactivators, the first and best characterized, counts among its members the steroid receptor coactivator-1 (SRC-1; Ref. 14); the transcriptional intermediary factor-2 [TIF-2, also known as glucocorticoid receptor-interacting protein-1 (15, 16) and SRC-2] and the coactivator amplified in breast cancer-1 (9), also known as SRC-3, receptor associated coactivator-3 (RAC-3), and p300/CBP/cointegrator associated protein (p/CIP) (17, 18). They share a 40% sequence homology primarily in the N terminus, which contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. TIF-2 is able to bridge amino and carboxyl termini of nuclear receptors, interacting at both activator function-1 and -2 domains to facilitate transcriptional activity (19).

It has been shown that TRß knockout mice (TRß-/-) have resistance to thyroid hormone (RTH; Refs. 20, 21, 22, 23, 24), whereas mice with disruption of the TR{alpha} 1 and 2 isoforms (TR{alpha}0/0) are hypersensitive to TH in several of the tissues examined (25). On the other hand, mice completely deficient in both TRß and TR{alpha} exhibit more severe resistance to TH than those lacking TRß only (26, 27). Taken together, these data suggest that both isoforms play selective and overlapping roles, both centrally and peripherally. Furthermore, coactivators are important in TR-mediated TH action in vivo as demonstrated by a mouse model with disruption of the SRC-1 gene (SRC-1-/-), which has also been shown to produce a phenotype of reduced ligand sensitivity (28, 29). Therefore, we ask whether SRC-1 differentially modulates the functions of TR{alpha} and TRß, and if so, how does this effect influence TH action and regulation of TSH in the pituitary?

We generated mice that were deficient in TR{alpha} and SRC-1 (TR{alpha}0/0SRC-1-/-), as well as mice deficient in the TRß and SRC-1 (TRß-/-SRC-1-/-) and compared with wild-type (WT) mice or mice with deficiency of either TR or SRC-1 alone. From these studies, we conclude that 1) TRß-/-SRC-1-/- mice demonstrate more severe TH resistance than either the SRC-1-/- or TRß-/- mice; the additive effect indicates that SRC-1 has an independent role in TH action over that of TRß and may enhance the activity of TR{alpha}; 2) SRC-1 facilitates TRß and TR{alpha}-mediated down-regulation of TSH, as TR{alpha}0/0SRC-1-/- mice demonstrate TH resistance rather than hypersensitivity as seen in TR{alpha}0/0mice; and 3) a compensatory increase in SRC-1 expression is associated with the TH hypersensitivity seen in TR{alpha}-deficient animals.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Baseline Thyroid Function Tests
Resistance to TH in the pituitary is defined as lack of suppression of serum TSH in the presence of elevated TH levels. The higher the TH levels without suppression of TSH implies a more severe state of impaired feedback or resistance. On the other hand, hypersensitivity to TH in the pituitary is defined as either normal or suppressed serum TSH in the presence of low or normal TH levels. Although baseline thyroid function tests can demonstrate both resistance and hypersensitivity to TH, hyporesponsiveness, or hyperresponsiveness to exogenous TH may also indicate resistance and hypersensitivity, respectively. Thyroid function tests in untreated mice of various genotypes are shown in Table 1Go. Experiments were performed and comparisons made in adult male mice because of previously reported sex and age differences in TH levels in WT mice (30, 31). Serum total and free T4 and T3 levels, as well as TSH concentrations, were significantly higher in TRß-/- and SRC-1-/- as compared with WT mice as previously reported (22, 23, 28). TR{alpha}0/0 mice demonstrated decreased serum T4 with normal TSH compared with WT mice (P < 0.005). Marked resistance to TH was observed in the combined TRß-/-SRC1-/- mice as demonstrated by serum levels of T4, T3, and TSH that were 1.7, 2.7, and 2.8 times greater, respectively, than in the resistant TRß-/- or 2.7, 2.8, and 10 times greater, respectively, than in the resistant SRC-1-/- mice. Deletion of both SRC-1 and TR{alpha} abrogated the hypersensitivity to TH seen in the TR{alpha}0/0, and at baseline the TR{alpha}0/0 SRC-1-/- mice had T4 and T3 values that were 1.4 and 1.2 times greater, respectively, than those of SRC-1-/- mice, whereas TSH values were not different. Levels of free T3 and T4 showed similar differences (Table 1Go).


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Table 1. Baseline Thyroid Function Tests in Male Mice of Different Genotypes

 
Effect of TH Deprivation and TH Treatment on Serum TSH and TSHß mRNA Expression in the Pituitary
TH deprivation was induced by a low iodine diet containing propylthiouracil (LoI/PTU). Thyroid function tests were evaluated after 14 d (Fig. 1Go). After LoI/PTU treatment, mice in each genotype had T4 levels below the level of detection (<0.25 µg/dl). Serum T3 values were suppressed by more than 50% of baseline and were not different among any group (Fig. 1Go compared with Table 1Go baseline values). All genotypes exhibited substantial increases in serum TSH levels in response to TH deprivation, i.e. LoI/PTU diet. (Fig. 1Go, shaded bars; compared with baseline values in Table 1Go). Serum TSH concentrations reached similar values in WT and SRC-1-/- mice (9,312 ± 888 and 9,276 ± 840 mU/liter, respectively). TRß-/- mice reached intermediate levels of TSH increase (14,926 ± 1,306 mU/liter) and TRß-/-SRC-1-/- mice had the highest TSH values, more than double those of WT animals (23,468 ± 1,440 mU/liter). Surprisingly, in the absence of TR{alpha} (either in the presence or absence of SRC-1) serum TSH values only increased to 50% of levels seen in WT or SRC-1-/- mice (TR{alpha}0/0; 4,754 ± 530 mU/liter; TR{alpha}0/0SRC-1-/-, 5,258 ± 594 mU/liter).



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Figure 1. Effects of TH Deprivation and T3 Treatment on Serum TSH Concentrations

Black bars are data after 14 d on LoI/PTU diet, and gray bars are after 4 d of 0.8 µg L-T3/100 g body weight·d while receiving the LoI/PTU diet. Each point is the mean ± SE and number of mice tested (N) is shown below each genotype. Relevant table of P values determined by Fisher’s protected least significant difference test is below the graph. Serum T4 and T3 concentrations, mean ± SE, are shown for mice after 14 d of LoI/PTU diet.

 
The influence of exogenously administered L-T3 to suppress the serum TSH of TH deprived mice was studied in all genotypes (Fig. 1Go, gray bars). WT and TR{alpha}0/0 mice demonstrated near complete suppression of serum TSH with L-T3 (46 ± 21 and 32 ± 10 mU/liter, respectively). Mice deficient in SRC-1, as previously demonstrated, had mild resistance to TH, and the additional loss of TR{alpha} (TR{alpha}0/0SRC-1-/-) did not result in any greater degree of resistance to TH when assessed by response to exogenous administration of TH. However, the TRß-/-SRC-1-/- mice were the most resistant to TH as demonstrated by suppression of serum TSH after L-T3 treatment to 12,645 ± 1,290 mU/liter. Moderate resistance to suppression was observed in the absence of the TRß with suppression of serum TSH after L-T3 treatment to 2,941 ± 308 mU/liter. The difference in TSH suppression among the different genotypes was not due to accelerated metabolism of administered L-T3, as serum concentrations achieved at different times after the administration of L-T3 were not significantly different among the animals of each genotype, either with single deletions (25) or in combination (data not shown). Furthermore, the lower mean values of T3 in TR{alpha}0/0SRC-1-/- as compared with TRß-/-SRC-1-/- contrasts with the greater degree of TSH suppression by T3 in the TR{alpha}0/0SRC-1-/- mice.

Effect of TH Deprivation and TH Treatment on TSH mRNA Expression in the Pituitary
Measurement of TSHß mRNA expression in the pituitary paralleled the changes observed in serum TSH values (Table 2Go). Expression of TSHß mRNA was increased more than 5,000-fold in response to TH deprivation in all genotypes examined with the greatest increase measured in the TRß-/- mice (20,930 ± 4,733-fold) and TRß-/-SRC-1-/- mice (55,476 ± 6,996). Treatment with TH in all genotypes resulted in a decrease in expression of TSHß mRNA related to untreated WT mice. However, TRß-/-, SRC-1-/-, TRß-/-SRC-1-/-, and TR{alpha}0/0SRC-1-/- had significantly less suppression, whereas the TR{alpha}0/0 had greater suppression compared with WT mice.


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Table 2. TaqMan Measurement of TSHß Expression in Pituitary

 
Expression of TSHß mRNA varied in mice of different genotypes at baseline and after TH deprivation. Therefore, to compare the effect of TH treatment, response of TSHß expression was made separately for each genotype relative the expression during TH withdrawal in that genotype. This analysis also showed that the TRß-/-, SRC-1-/-, TRß-/-SRC-1-/-, and TR{alpha}0/0SRC-1-/- were relatively more resistant to TH suppression compared with the WT mice and that the TR{alpha}0/0 were more sensitive to TH. Whereas the absence of either TR{alpha} or SRC-1 also conferred either no resistance or mild resistance, respectively, absence of both TR{alpha} and SRC-1 resulted in a phenotype similar to the TRß-/-SRC-1-/-.

Expression of TR Isoforms in the Pituitary
To determine if deletion of TR or coactivator resulted in changed expression of the remaining TR isoforms we measured mRNA expression ({alpha}1, {alpha}2, ß1, and ß2) for each genotype in pituitary glands at baseline (Fig. 2Go, A–D). Values are expressed relative to the mean level of pituitary glands from WT mice for each experiment. The most striking finding is that deletion of SRC-1 alone, or deletion with TR{alpha} or TRß always up-regulates the TRs. SRC-1-/- mice had the trend for an increase in TRß2 (2.5-fold; P = 0.06) and a robust increase of TRß1 (8.6-fold, P < 0.0001). TR{alpha}0/0SRC-1-/- mice had a 3-fold increase in TRß1 expression and a 4-fold increase of TRß2 expression over WT (Fig. 2Go, A and B). Whereas genotype had no significant effect on TR{alpha}2 expression (Fig. 2CGo), there was a 10-fold and 5-fold increase in TR{alpha}1 expression in TRß-/-SRC-1-/- and SRC-1-/- mice, respectively (Fig. 2DGo).



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Figure 2. TR Isoform mRNA Expression in the Pituitary Gland at Baseline

mRNA expression is shown as fold change relative to WT mice ± SE as determined by TaqMan quantitative real-time PCR. All animals are on a regular chow diet without TH treatment. Number of animals in each group is shown below the genotype (N). Results are shown for TRß2 (A), TRß1 (B), TR{alpha}2 (C), and TR{alpha}1 (D). Asterisks identify P values for differences between WT animals and other genotypes as determined by Student’s t test; *, P <= 0.05; **, P = 0.0005; ***, P < 0.0001.

 
Removal of TR{alpha} or TRß had no or little effect on the other TRs. TR{alpha}0/0 mice showed no significant increase in TRß2 and a 2.7-fold increase in TRß1. TRß-/- had no change in expression of either TR{alpha}1 or TR{alpha}2. These data suggest that there is little reciprocal regulation of TR{alpha} and TRß gene expression in the pituitary.

The specificity of the TaqMan reaction was confirmed for TR{alpha}1 and TR{alpha}2 by using pituitary extracts from TR{alpha}0/0 mice lacking both TR{alpha}1 and TR{alpha}2 and demonstrating appropriate linear dilution of TR{alpha}-containing WT pituitary RNA extracts (Fig. 3Go). Values of TR{alpha} mRNA less than 20% of WT were off the curve and considered undetectable. All levels of TR{alpha} measured were greater than or equal to WT (>= 1, or 100%), confirming the sensitivity of the assay. Primers used for detection of TRß isoforms were targeted at a region upstream from the area of gene disruption, and therefore we could not measure TRß1 and TRß2 specific mRNA expression in TRß-/- mice.



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Figure 3. Specificity of Probes for TR{alpha}1 and TR{alpha}2

After reverse transcription of mRNA using 10 µg total RNA from WT, 10 ng cDNA was used for serial dilution with cDNA from TR{alpha}o/o mice. Results are expressed as percentage of undiluted mRNA concentration. Points are the mean of duplicate samples.

 
In Vitro Binding of T3 to Pituitary Nuclei
We investigated whether the observed increase in expression of TRß2 in the TR{alpha}0/0, SRC-1-/-, and TR{alpha}0/0SRC-1-/- resulted in a significant increase of T3 binding to the nuclei of pituitary in these mice. Specific T3 binding was measured from nuclei isolated from three pituitaries of mice of each genotype (Tables 3Go and 4Go). Because the number of nuclei isolated from pituitaries were limited, Scatchard analysis was not performed, therefore, maximal binding capacity (MBC) was estimated assuming the association constant (Ka) of the TR expressed in the pituitary of each genotype was the same as the Ka for TRs in liver (5.5 x 109 M-1). In TRß-/- mice, the MBC was 58% of WT, consistent with observations that TRß is the predominant TR expressed in the pituitary. In the TR{alpha}0/0, SRC-1-/-, and TR{alpha}0/0SRC-1-/-, the MBC/pituitary relative to WT correlated with the quantitation of TRß2 mRNA expression determined by quantitative PCR analysis (Table 4Go). In TR{alpha}0/0SRC-1-/- mice, although the MBC/pituitary was greater than WT, it was not as high as that predicted from the real-time PCR expression of TRß2.


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Table 3. Measurement of MBC and DNA in Pituitaries of Different Mice

 

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Table 4. Relative Amounts of TRß2 Expression and T3 Binding in Nuclei from Pituitaries of Mice of Different Genotypes Compared with WT

 
It was noted that there is an increase in the number of nuclei observed in the experimental genotypes as determined by the amount of DNA/pituitary (Table 3Go) suggesting pituitary hyperplasia in these mice.

Coactivator Expression in the Pituitary
Levels of three coactivator, SRC-1, TIF-2, and SRC-3 mRNAs were measured in RNA extracted from pituitaries of individual, untreated mice of different genotypes by quantitative PCR. In TR{alpha}0/0 mice, SRC-1 mRNA was 4-fold increased over that of WT (P < 0.0006; Fig. 4AGo). There was no SRC-1 expression in SRC-1-deficient animals. This was confirmed by performing a dilution curve of pituitary mRNA taken from SRC-1-expressing animals and showing a linear 1:1 serial dilution of mSRC-1 levels with mRNA from SRC-1-/- pituitaries down to 2.5 ng sensitivity, considered to be background noise in this assay (Fig. 4BGo).



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Figure 4. SRC-1, TIF-2, and SRC-3 Coactivator mRNA Expression in the Pituitary and TaqMan Probe Specificity

Data are shown as fold change relative to WT mice ± SE as determined by quantitative real-time PCR. All animals are on a regular chow diet. Number of animals in each group is shown below the genotype (N). Results are shown for SRC-1 (A), TIF-2 (C), and SRC-3 (E). The bottom panels depict specificity of TaqMan probes. After reverse transcription of mRNA using 10 µg total RNA from WT, 10 ng cDNA was used for serial dilution with cDNA from respective knockout mice: SRC-1 (B), TIF-2 (D), and SRC-3 (F). Results are expressed as percentage of undiluted mRNA concentration. Points are the mean of duplicate samples. Asterisks identify P < 0.05 for differences between WT animals and other genotypes as determined by Student’s t test.

 
Expression of the p160 family member TIF-2 was analyzed in the same animals. There was a 3.5-fold increase in TIF-2 expression over WT (P = 0.0002) in TRß-/-SRC-1-/- mice (Fig. 4CGo). Increases in TIF-2 expression in TRß-/-SRC-1-/- animals were significantly greater than all other genotypes. In addition, fidelity of this probe was analyzed as for the SRC-1 probe by using mRNA taken from pituitaries of TIF-2-/- animals (Fig. 4DGo). The TIF-2-/- mouse was obtained by deletion of exonic sequences containing the NR boxes (nucleotides 1330–2597, accession no. U39060 of TIF-2), resulting in a null mutation.

The third member of the p160 coactivator family, SRC-3, was investigated. Levels of SRC-3 mRNA were unchanged in animals lacking the TR{alpha} or TRß (Fig. 4EGo). In the absence of SRC-1, animals showed increased expression of SRC-3, with levels of expression 2.6-fold higher than WT (P = 0.0001). In addition, in the absence of both SRC-1 and each of the TR{alpha} and TRß, levels of SRC-3 were also significantly higher than WT (P = 0.03; P = 0.02, respectively). Specificity of the SRC-3 probe was determined using RNA derived from liver of SRC-3-/- animals (Fig. 4FGo). SRC-3-/- mice were created by deletion of exons 2–13 (amino acids 11–891, accession no. NM008679 of the SRC-3), resulting in a null mutation.

The influence of thyroid status on expression of SRC-1, TIF-2, and SRC-3 mRNA in the pituitary of WT mice was investigated (Table 5Go). There was a small decrease in TIF-2 and SRC-3 coactivator mRNA expression in hypothyroid WT mice compared with untreated WT mice. However, treatment of hypothyroid mice with T3 resulted in a 2.5-fold increase in SRC-3 expression and a slight but significant increase in SRC-1 and TIF-2 (Table 5Go).


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Table 5. Coactivator mRNA Changes with TH Deprivation and Treatment in WT Mice

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SRC-1 Interacts with both TR{alpha} and TRß pathways during states of TH withdrawal and TH treatment.

The importance of SRC-1 in regulating TH action in the pituitary has been previously demonstrated in the SRC-1-/- mice, which showed reduced sensitivity to TH (28). However, it was not known whether the effects of SRC-1 on TH-regulated gene transcription were mediated through specific TR subtypes. In this study, thyroid function tests were compared in mice that were deficient in SRC-1 with or without deletion of TR{alpha} or TRß. Mice deficient in both the TRß and SRC-1 have profound TH resistance as demonstrated by markedly elevated serum TSH concentrations in the presence of substantially increased serum levels of T4 and T3 over WT. This degree of resistance exceeds that seen in mice deficient in the TRß-/- (23) and SRC-1-/- alone (28). Evaluation of serum TSH concentration reveals values similar to those of TR{alpha}0/0/TRß-/- double knockout mice (22, 27, 30) and suggests that SRC-1 interacts with both TR{alpha} and TRß. Additional evidence for a synergistic effect of TRß and SRC-1 deficiency resulting in profound resistance to TH is the result of exogenous L-T3 treatment on serum TSH concentration. These data indicate that SRC-1 acts at the thyrotroph to mediate TH action via the TRß. The fact that disruption of both SRC-1 and TRß has an additive effect, with a more profound resistance than in TRß-/- animals, demonstrates that SRC-1 is involved in a parallel, redundant receptor pathway, most likely with TR{alpha}.

Our laboratory previously reported that mice deficient in TR{alpha} are hypersensitive to TH, in that they have significantly lower serum free T4 index without significant change in TSH compared with WT littermates (25). In the current report, TR{alpha}0/0SRC-1-/- mice do not demonstrate hypersensitivity to TH but at baseline have higher serum TH levels than SRC-1-/- mice with similar TSH levels. In addition, TH deprived TR{alpha}0/0SRC-1-/- mice, when treated with TH, suppress serum TSH to similar levels as those seen in SRC-1-/- mice. The baseline TH levels indicate that the TR{alpha} may play a role in the suppression of TSH by TH, and that this function is mediated by SRC-1, as TR{alpha}0/0SRC-1-/- mice have higher TH levels than either the SRC-1-/- or the TR{alpha}0/0 mice individually. Therefore, SRC-1 and TR{alpha} have complementary effects, which strongly suggest that SRC-1 also works through a redundant pathway with TRß. We ruled out a possible effect of different L-T3 metabolism in the various genotypes.

In animals made hypothyroid with a LoI/PTU diet, all showed marked up-regulation of serum TSH levels as well as TSHß mRNA expression in the pituitary. Interestingly, TRß-/-SRC-1-/- mice had a more robust increase in serum TSH than any of the other genotypes. On the other hand, animals deficient in TR{alpha} (both TR{alpha}0/0 and TR{alpha}0/0SRC-1-/-) have increased serum TSH levels when deprived of TH, yet they achieve only half the levels of WT mice, regardless of the presence or absence of SRC-1. This would indicate that the presence of the TR{alpha} facilitates the release of TSH in response to TH deprivation. Interestingly, the level of enhancement of serum TSH as well as TSHß expression is the same in TR{alpha}0/0 and TR{alpha}0/0SRC-1-/- mice, which suggests that TRß, which is the only TR present in these mice, does not need SRC-1 to activate TSH in these conditions. An alternative explanation is that, because there are small amounts of T3 present in the LoI/PTU-treated animals, and whereas this small amount is not suppressive of serum TSH in the presence of TR{alpha}, it is able to prevent a maximal increase in serum TSH levels when TRß is the only isoform present. The latter explanation seems likely because deficient in both TR{alpha} and TRß (TR{alpha}0/0/TRß-/-) demonstrate serum TSH levels that are higher than those observed in TRß-/- mice (22). Serum TSH measurement reflects the transcription of TSHß mRNA in the thyrotroph. Other investigators have shown a 10-fold increase in TSHß mRNA in TRß-/-, consistent with the elevated serum TSH (20, 32). We have also demonstrated a more than 5000-fold increase in TSHß mRNA expression in all genotypes compared with untreated WT mice. The lesser increase in serum TSH in TR{alpha}0/0 mice compared with WT was not observed in TSHß mRNA expression, suggesting that the latter may be a posttranslational effect in the TR{alpha}0/0 mice.

Increase in TRß2 Expression and T3 Nuclear Binding Is Insufficient to Overcome the Resistance to TH
The increase in TRß2 expression measured by real-time PCR generally correlated with the increased MBC measured in isolated pituitary nuclei. However, the increased MBC is not sufficient to overcome the resistance to TH that has been demonstrated in these mice indicating a role for SRC-1 to realize the full effect of TH action. The increased sensitivity to TH in the TR{alpha}0/0 mice may in part be due to an increase in TRß2 expression and increased T3 binding (see below).

SRC-1 Aids TRß-Mediated Repression, not Activation of TSH Gene Expression in the Presence of TH
During TH deprivation, we observed the greatest increase in serum TSH in the TRß-/-SRC-1-/- mice, where only TR{alpha} is present. This indicates the importance of TR{alpha} and SRC-1 interaction in the absence of TH. Similar to TH withdrawal, during TH treatment there is a strong interaction of TR{alpha} and SRC-1 as the TRß-/-SRC-1-/- mouse there is minimal suppression of TSH. The term "interaction" implies not only a direct physical interaction between TR and SRC-1 but also includes reference to interaction among SRC-1 and other transcription factors that may influence the transcriptional activity of TR.

The interaction of TRß and SRC-1 can be assessed by comparing the TR{alpha}0/0 and TR{alpha}0/0SRC-1-/- mice. During TH withdrawal, the absence of SRC-1 does not influence release of TH, whereas during TH treatment the absence of SRC-1 impairs the ability of TRß to suppress TSH, indicating that SRC-1 mediates TRß suppression by TH and not activation.

The mechanism of down-regulation of TSH expression in the presence of TH is not well understood (33). TH, similar to steroid hormones and retinoids, regulates gene expression by binding nuclear receptors and recruitment of coactivators which posses intrinsic HAT (13). The HAT activity is essential for RNA polymerase initiation of gene transcription. However activation of the TR with TH turns off TSH transcription. Xu et al. (34) have shown that coactivator-associated arginine methyltransferase 1 can methylate the class of p160 coactivators. This results in loss of HAT activity and conversion into a corepressor. The methylation of SRC-1 by coactivator-associated arginine methyltransferase 1 may thereby be a mechanism for TRß/SRC-1 interaction during negative regulation of TSH with TH. In fact, it may be the interaction of SRC-1 with other transcription factors regulating TSH expression, which contribute to the physiological events observed. For example, one cannot exclude the involvement of retinoid X receptor, and other transcription factors, which may be involved in SRC-1-mediated TH action.

The interaction between SRC-1, TRß, and TR{alpha} in regulation of thyrotroph TSH expression is summarized in the model in Fig. 5Go. In summary, the interaction of SRC-1 and TRß is stronger in the presence of TH, and the functional interaction of SRC-1 with TR{alpha} is stronger in the absence of TH. It should be noted that inherent in this model is that the absence of SRC-1 and TRs in our model system are not tissue selective, and one is unable to determine what adaptive processes have occurred during pituitary development that contribute to the final phenotype observed.



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Figure 5. Model for Interaction of SRC-1 with TR{alpha} and TRß in Regulation of Thyrotroph TSH Expression

The shaded upper panels represent events in the absence of TH, when serum TSH increases, whereas the lower panels represent events in the presence of TH where there is a decrease in serum TSH. See Discussion for details.

 
Pituitary Expression of Other p160 Coactivators Is Increased in the Absence of SRC-1
Many of the coactivators are expressed in multiple tissues, suggesting that there is either redundancy in their function or receptor complex specificity. Deletion of SRC-1 resulted in 2-fold increases in mRNA expression of the coactivator TIF-2 as measured by Northern blot in brain and testes (29). Currently, we demonstrate the same up-regulation of TIF-2 (2-fold) in the pituitary of SRC-1-/- mice by quantitative real-time PCR. Furthermore, a 3.5-fold increase in TIF-2 expression in pituitaries of TRß-/-SRC-1-/- mice over WT was shown. Additionally, we report significantly increased pituitary expression of the p160 coactivator, SRC-3, in SRC-1-/-; TR{alpha}0/0SRC-1-/-; and TRß-/-SRC-1-/- mice (2.6-, 2.1-, and 1.6-fold, respectively). The increase in SRC-3 expression is independent of receptor subtype and is only effected by the loss of SRC-1. Additionally, SRC-3 expression is increased in response to T3, which in part may be responsible for its increased expression in mice deficient in SRC-1, as these mice have higher TH levels. These data demonstrate that, in the absence of SRC-1, coactivators in the p160 family, specifically TIF-2 and SRC-3, increase mRNA expression in the pituitary, yet this increased expression does not obviate the RTH seen in TRß-/-, SRC-1-/-, and TRß-/-SRC-1-/- mice. These data, however, cannot rule out the possibility that the degree of resistance to TH would not be more severe in the absence of a combination of these coactivators (35). Although these coactivators have been shown to have interactions with individual TRs in vitro (36), these data are the first to demonstrate such an interaction between distinct coactivators and TRs in vivo. An important next step would be to investigate direct interaction between coactivators and TR subtypes in vitro, though isolating a cell line devoid of all coactivators to test these specific interactions is difficult, as these coactivators are ubiquitously expressed, as mentioned above, and they interact with many molecules. To use an in vitro system would require not only isolation of individual coactivators but also require depletion of other receptors that interact with them. Therefore, we feel as though the in vivo approach, which acknowledges the additional interactions of other factors in the cell, is a less biased approach.

Increase in SRC-1 Expression Contributes to the Hypersensitivity to TH in TR{alpha}0/0 Mice
SRC-1 mRNA expression levels in pituitaries of TR{alpha}0/0 mice increased 4-fold over WT animals. SRC-1a compared with the SRC-1e isoform was previously reported to be higher in rat pituitary by in situ hybridization (37); however, we were unable to distinguish between SRC-1a and 1e isoforms (38) due to the design of the primers for the PCR. Because the hypersensitivity of the TR{alpha}0/0 mice is abrogated by simultaneously removing SRC-1 (TR{alpha}0/0SRC-1-/- mice) as determined by baseline TH levels, then the increase in SRC-1 mRNA levels, if accompanied by increases in pituitary SRC-1 protein levels, may account for the hypersensitivity phenotype previously reported in TR{alpha}0/0 mice (25). Increased TH sensitivity would be due to greater availability of the SRC-1 to interact with the TRß and facilitate TH action, as has been shown previously in vitro (35). It had been hypothesized that the absence of the TR{alpha}2 was most likely responsible for mediating the hypersensitivity seen in TR{alpha}0/0 mice. The latter was thought to be due to an inhibitory role TR{alpha}2 plays by its inability to bind the T3 ligand yet still bind to the TRE.

Although the current data do not rule out the contribution of TR{alpha}2, it may be less important than the effect of increased SRC-1 levels, as TR{alpha}0/0SRC-1-/- mice have moderate RTH, negating the hypersensitivity phenotype seen in TR{alpha}0/0 mice discussed above. Furthermore, it is possible that the expression of SRC-1 is directly regulated by TR{alpha}2. One could answer this question by studying mice with TR{alpha}2-specific deletion. Recently, a mouse with selective disruption of TR{alpha}2 has been reported with a mixed phenotype of hypo and hyperthyroidism; however, overexpression of TR{alpha}1 in this mouse prevents its use as a model to test this hypothesis (39). A TR{alpha}2 deletion cannot be produced in dissociation of its effect on TR{alpha}1. Therefore, in the pituitary, SRC-1 action mediating TH-regulated transcription appears to be TR isoform specific. Whether the role SRC-1 plays in other tissues to mediate TH action is also TR isoform specific is under investigation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation and Handling of Animals
Mice deficient in SRC-1 (SRC-1-/-) were generated by a targeting vector that disrupted the SRC-1 gene in 129 sv embryonic stem cells that inserted an in-frame stop codon at the Met-381 position and deletion of approximately 9 kb of downstream genomic sequence that contains 446 amino acids from Met-381 to Thr-826. This eliminated all functional domains for transcriptional activation, HAT activity and interactions with nuclear receptors, cAMP response element binding protein-binding protein, p300, and p/CAF (29). The SRC-1-/- construct was maintained in a C57BL/6J mouse strain. The genotype of mice was confirmed by analysis of tail DNA as previously described (29).

The TRß-/- mice were produced by insertion of the LacZ-NeoR cassette downstream to the splice site in exon 4, eliminating the expression of the DNA and ligand binding domains of TRß1 and TRß2 (22). The TR{alpha}0/0 mice were produced by insertion of the LacZ-NeoR cassette downstream from exon 3 and replacing exons 5–7. This effectively abolished not only the generation of TR{alpha}1 and TR{alpha}2 transcripts, but also that of TR{Delta}{alpha}1 or TR{Delta}{alpha}2 by removing a transcription start site in intron 7 (27). The gene sequence for rev-erbA {alpha} protein encoded by the opposite strands for the TR{alpha} (40) remains intact. In both sets of mice, the recombinant embryonic stem cells were derived from 129 sv mice and were implanted into C57BL/6 recipient blastocysts. C57BL/6 mice were mated to each chimeric mouse and then backcrossed at three to four times into the same strain, thereby diluting the 129 sv background.

The SRC-1-/- mice were crossed with TR{alpha}0/0 and TRß-/- mice to produce double heterozygous animals. These animals were backcrossed more than five times on a C57BL/6 background to produce a uniform genetic background for WT and combination knockout animals.

Mice were weaned on wk 4 after birth and were fed Purina Rodent Chow (0.8 ppm Iodine; Purina Mills, St. Louis, MO) ad libitum and tap water. They were housed, 5 or less mice per cage, in an environment of controlled 19 C temperature and 12 h alternating darkness and artificial light cycles. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at the University of Chicago.

Mice were 40–70 d old at the time they were killed. At various intervals, approximately 300 µl of blood were obtained by tail vein under light methoxyflurane (Pitman Moore, Mundelein, IL) anesthesia. Experiments were terminated by exsanguination via retroorbital vein. Whole blood was allowed to clot overnight at 4 C, and serum was collected after centrifugation and stored at -20 C until analyzed.

Induction of Hypothyroidism and Treatment with TH
TH deficiency was induced in male mice by feeding with low iodine (LoI) diet supplemented with 0.15% propylthiouracil (Harlan Teklad, Madison, WI). On d 11, groups (3–6 mice each) of each genotype were injected once daily for 4 d with the vehicle only (1x PBS) and others received 0.8 µg of L-T3/100 g body weight·d, maintained on the LoI/PTU diet. Fourteen to 16 h after the final injection, experiments were terminated by exsanguination. L-T3, dissolved in PBS and 0.002% human serum albumin as a vehicle, was given by ip injection in a total volume of 0.1–0.3 ml. A stock of L-T3 (Sigma, St. Louis, MO) at a concentration of 1 mg/ml was prepared in a solution of 50% ethanol/50% 1x PBS containing 5 mM NaOH and kept at -20 C, protected from light. Concentration of L-T3 was confirmed by RIA (Diagnostic Products Corp., Los Angeles, CA). Blood samples were obtained at baseline, on d 10 after the initiation of the LoI/PTU diet and at the termination of the experiment on d 14.

The dose of L-T3 given to thyroid hormone deficient animals was derived from previous experiments. It was optimized to achieve a partial suppression of serum TSH in order to make evident the differences between WT and SRC-1-/- mice (25, 28). Metabolism of T3 was determined in each genotype by measuring serum T3 levels at 2, 4, 8, and 16 h following injection of L-T3 (25).

Measurements of TH and TSH Concentrations in Serum
Serum TSH was measured in 50 µl of serum using a sensitive, heterologous, disequilibrium double antibody precipitation RIA as previously described (31). Samples containing more than 200 mU TSH/liter were 5- and 50-fold diluted with a TSH-deficient mouse serum.

Serum T4 and total T3 concentrations were measured by a double antibody precipitation RIA (Diagnostic Products Corp.) using 25 and 50 µl of serum, respectively. The sensitivity of these assays were 0.2 µg T4/dl and 5 ng T3/dl. The interassay coefficients of variation were 5.4, 4.2, and 3.6% at 3.8, 9.4, and 13.7 µg/dl for T4 and 7.7, 7.1, and 6.2% at 32, 53, and 110 ng/dl for T3.

Isolation of Pituitary RNA
Pituitaries from animals were immediately frozen on dry ice and stored at -80 C. For RNA extraction, individual pituitaries were homogenized in 100 µl of TRIzol (Life Technologies, Inc., Rockville, MD) with a battery-operated, hand-held device, and total RNA was extracted according to the protocol provided with the TRIzol reagent. Concentration (A260) of the total RNA was determined, and RNA was stored in ethanol at -80 C.

TaqMan RT-PCR of TRs, SRC-1, TIF-2, SRC-3, and TSHß in Pituitary
To quantitate mRNA expression of various genes in pituitaries of different genotypes, 2 µg of total RNA was reverse transcribed using the First-Strand Synthesis Superscript Kit (Life Technologies, Inc., Rockville, MD) according to the provided protocol. Reverse transcription was performed using random hexamers. cDNAs obtained from the reverse transcription reaction were diluted with ribonuclease-free water to a concentration of 1 ng/µl. TaqMan fluorescent probe/primer sets were designed using Primer Express 1.5 (PE Applied Biosystems, Foster City, CA) and mRNA sequences taken from GenBank. Specificity was confirmed by BLAST search. Primer/probe sets were then obtained for mouse (m)TR{alpha}1, mTR{alpha}2, mTRß1, mTRß2, mSRC-1, mTIF-2 mSRC-3, and mTSHß (MegaBases, Evanston, IL; Table 6Go). Primers were designed to detect the area deleted in the SRC-1-/-, TR{alpha}0/0, SRC-3-/- and TIF-2-/- mice. Isoforms produced by the TRß gene form from differences in the N termini of the transcription products. Therefore, in the design of the TRß-/- mice, disruption of the TRß gene was made after the start site for all isoforms. As they differ only in their N termini, probes specific for each TRß isoform had to be designed upstream of the knockout construct in the TRß-/- mice. For this reason, animals showed N-terminal expression of the TRß, although there is no expression of the protein in these animals (22). Primers and probes made were specific for either TRß1 or TRß2. Equal loading of wells was controlled using a commercially available probe/primer set for 18S ribosomal RNA (PE Applied Biosystems). Detection of mRNA was performed with Sequence Detector Software and ABI 7700 Sequence Detection System (PE Applied Biosystems), capable of reading two fluorophores simultaneously (6-carboxy-fluorescein-specific probe and VIC-Ribosomal control). Ten nanograms of reverse-transcribed cDNA sample were run in each well in duplicate and the reaction was performed using TaqMan Universal Mix and 96-well optical plates (PE Applied Biosystems) with each primer at a final concentration of 0.3 µM. Each duplicate sample represents reverse transcribed total RNA from an individual mouse pituitary. The threshold cycle (Ct) is the first cycle, in a 40 cycle reaction, at which fluorescence is detected. For each sample, there are two recorded Cts, the first corresponding to amplification of 18S rRNA (VIC fluorophore), and the second to specific gene of interest (6-carboxy-fluorescein fluorophore). Normalization of data involved subtraction of rRNA Ct from that of the specific gene being amplified per well. For each mouse genotype analyzed, at least five individual pituitary RNAs were run in duplicate. To calculate results, the average normalized Ct for WT mice was determined. Individual mouse pituitary data were reported as a fold increase or decrease from this WT average. Assays were repeated at least three times, and the data were normalized and merged. Because multiple assays were performed, the WT were compared with each other and are reported as 1 with a SE.


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Table 6. Probe/Primer Sets for TaqMan Quantitative Real-Time PCR Used for mRNA Quantitation

 
TSHß mRNA expression was also determined using the method as described above, except instead of a specific fluorescent probe, the QuantiTest SYBR Green PCR Kit (QIAGEN, Valencia, CA) was used. Data were normalized to 18S RNA on the identical sample run in parallel.

Measurement of Nuclear T3 Binding
Three pituitaries obtained from untreated mice of each genotype were combined and nuclei were isolated as previously reported (24). Isolated nuclei from each genotype were suspended in 1 ml of SMTDP buffer [0.32 M sucrose, 1 mM MgCl2, 20 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride] and divided into two glass tubes. Nuclei were incubated for 2 h at 25 C with 5 x 10-10 M [125I]T3 (S. A. 81.4 TBq/mmol, NEX110X, NEN Life Science Products, Boston, MA) with or without 3 x 10-7 M unlabeled T3 to determine nonspecific T3 binding. After the incubation, the nuclei were collected by centrifugation at 1700 x g for 10 min at 4 C and washed two times with SMTDP buffer plus 0.5% Triton X-100. Radioactivities in the pellets were counted by gamma scintillation counter (ARC-1000 M, Aloka, Tokyo, Japan) and DNA contents in the pellets were determined by Burton’s method (41). Release of nuclear TR was also measured as described previously (24); however, no significant release was observed in the present assay for pituitaries. By subtraction of the nonspecific T3 binding, specific T3 binding activity was determined. Because number of nuclei isolated from pituitaries was limited, we were not able to perform Scatchard analysis. So MBC was estimated assuming that the Ka of TR in pituitaries of each genotype was the same as that in liver (5.5 x 109 M-1) and expressed as fmol/pituitary.

Data Presentation and Statistics
Values are reported as mean ± SE, except for Fig. 2Go where the results are mean ± SD. P values were calculated using ANOVA, Fisher’s protected least significant difference test, or the Student’s t test where indicated. Values corresponding to the respective limits of the assays’ sensitivities were assigned to samples that measured below the detectable range.


    ACKNOWLEDGMENTS
 
The authors would like to thank Prof. Samuel Refetoff for his invaluable input, critical discussions, guidance, and support in the project. In addition, the authors wish to thank Graeme Bell for the use of his ABI7700 machine (PE Applied Biosystems) for TaqMan reactions and Prof. P. Chambon and Dr. M. Gehin for tissues from TIF-2-/- mice for use as blank in the quantitative PCR.


    FOOTNOTES
 
This work was supported in part by NIH Grants DK-58281 (to R.E.W.), HD-078587 (to B.O’M.), and DK-58242 (to J.X.); Ministry of Research Grant ACI 283 (to J.S.); Grant-in-Aid for Scientific Research (B) 14370324 (to Y.M.); Health and Labor Science Research Grant for Research on Specific Disease from Ministry of Health, Labor and Welfare (to H.S.); and by the Seymour J. Abrams Thyroid Research Center.

Abbreviations: Ct, Threshold cycle; HAT, histone acetyltransferase; Ka, association constant; LoI/PTU, low iodine diet containing propylthiouracil; m, mouse; MBC, maximal binding capacity; RTH, resistance to thyroid hormone; SRC-1, steroid receptor coactivator-1; TH, thyroid hormone; TIF-2, transcriptional intermediary factor-2; TR, TH receptor; TRE, TH response elements; WT, wild-type.

Received for publication May 9, 2002. Accepted for publication January 29, 2003.


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 MATERIALS AND METHODS
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