Specificity of thyroid hormone receptor subtype and steroid receptor coactivator-1 on thyroid hormone action

Peter M. Sadow1,2, Olivier Chassande3, Karine Gauthier3, Jacques Samarut3, Jianming Xu4, Bert W. O'Malley4, and Roy E. Weiss1

Departments of 1 Medicine and 2 Pathology, University of Chicago, Chicago, Illinois 60637; 3 Laboratoire de Biologie Moléculaire et Cellulaire de l'Ecole Normale Supérieure de Lyon, 69364 Lyon, France; and 4 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isoforms of the thyroid hormone receptor (TR)alpha and TRbeta genes mediate thyroid hormone action. How TR isoforms modulate tissue-specific thyroid hormone (TH) action remains largely unknown. The steroid receptor coactivator-1 (SRC-1) is among a group of transcriptional coactivator proteins that bind to TRs, along with other members of the nuclear receptor superfamily, and modulate the activity of genes regulated by TH. Mice deficient in SRC-1 possess decreased tissue responsiveness to TH and many steroid hormones; however, it is not known whether or not SRC-1-mediated activation of TH-regulated gene transcription in peripheral tissues, such as heart and liver, is TR isoform specific. We have generated mice deficient in TRalpha and SRC-1, as well as in TRbeta and SRC-1, and investigated thyroid function tests and effects of TH deprivation and TH treatment compared with wild-type (WT) mice or those deficient in either TR or SRC-1 alone. The data show that 1) in the absence of TRalpha or TRbeta , SRC-1 is important for normal growth; 2) SRC-1 modulates TRalpha and TRbeta effects on heart rate; 3) two new TRbeta -dependent markers of TH action in the liver have been identified, osteopontin (upregulated) and glutathione S-transferase (downregulated); and 4) SRC-1 may mediate the hypersensitivity to TH seen in liver of TRalpha -deficient mice.

knockout; resistance to thyroid hormone; thyrotropin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THYROID HORMONE RECEPTOR (TR)alpha and TRbeta function as nuclear transcription factors that mediate thyroid hormone (TH) action. TRs bind to TH response elements on thyroid-responsive genes in association with transcriptional coregulators. Corepressors form part of a transcriptional complex and recruit histone deacetylases, reducing transcription (18, 40). The conformational change of TR produced by TH binding releases the corepressor and permits the recruitment and binding of a coactivator. The coactivator has a number of functions that may include intrinsic histone acetyltransferase activity, recruitment of histone acetyltransferases, and recruitment of additional transcription factors and RNA polymerase (21, 23). Several classes of nuclear coactivators have been described that are important in mediating the response of mammalian cells to thyroid as well as steroid and retinoid hormones (2, 7, 14, 17, 41, 43), among which is the steroid receptor coactivator (SRC)-1, a member of the p160 family of coactivators (25).

Because some genes are upregulated and others are downregulated by TH in the same cell, various theories have been proposed to explain the mechanism(s) of TR-modulated gene expression (see Ref. 44 for review). We propose that specificity of interaction among TR subtypes with particular cofactors may influence whether there is stimulation or inhibition of mRNA expression. Determination of the nature of interaction of TR subtypes with specific cofactors, and how it affects gene transcription, can be evaluated in vivo by using animals that are lacking each of the two TR genes with or without SRC-1. It has been shown that TRbeta knockout mice (TRbeta -/-) have resistance to TH (10-12, 22, 34, 37), whereas mice with disruption of the TRalpha 1 and -alpha 2 isoforms (TRalpha 0/0) are hypersensitive to TH in several of the tissues examined (22) or less prone to the effects of TH deprivation (24). On the other hand, mice completely deficient in both TRbeta and TRalpha exhibit more severe resistance to TH than those lacking TRbeta only (16). 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 hormone sensitivity (38, 42). Therefore, we ask whether SRC-1 differentially modulates the functions of TRalpha and TRbeta , and if so, how this effect influences TH action in the liver and heart.

For this purpose, we generated mice deficient in TRalpha and SRC-1 (TRalpha 0/0SRC-1-/-), as well as mice deficient in TRbeta and SRC-1 (TRbeta -/-SRC-1-/-), and compared these with wild-type (WT) mice or mice with deficiency in TRbeta , TRalpha , and SRC-1 alone. Our data provide evidence that, in the absence of TRalpha or TRbeta , SRC-1 is important for normal growth. We also show that SRC-1 mediates TRalpha and TRbeta action in the heart. This study identifies two novel TRbeta -dependent markers of TH action in the liver, osteopontin (upregulated) and glutathione S-transferase (downregulated). We have also demonstrated that SRC-1 may mediate the hypersensitivity to TH seen in TRalpha 0/0 mice.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation and Handling of Animals

SRC-1-/- mice were generated as reported previously (42). A targeting vector disrupted the SRC-1 gene in 129sv ES cells by inserting an in-frame stop codon at the Met381 position and deleting ~9 kb of downstream genomic sequence that contains 446 amino acids from Met381 to Thr826. This eliminated all functional domains for transcriptional activation, histone acetyltransferase activity, and interactions with nuclear receptors CBP, p300, and p/CAF (42). 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 (42).

TRbeta -/- mice were produced, as previously described (12), 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 TRbeta 1 and TRbeta 2. The TRalpha 0/0 mice were produced by insertion of the LacZ-NeoR cassette downstream from exon 3 and replacing exons 5 through 7. This effectively abolished not only the generation of TRalpha 1 and TRalpha 2 transcripts but also that of TRDelta alpha 1 or TRDelta alpha 2 by removing a transcription start site in intron 7 (13). The gene sequence for rev-erbA-alpha protein encoded by the opposite strands for the TRalpha (31) remains intact. In both sets of mice, the recombinant ES cells were derived from 129sv mice and were implanted into C57BL/6 recipient blastocysts. C57BL/6 mice were mated to each chimeric mouse and then backcrossed 3-9 times into the same strain, diluting the 129sv background.

The individual knockout mice were backcrossed more than nine times on a C57BL/6 background to produce a uniform genetic background for WT and knockout animals. We then crossed the SRC-1-/- mice with TRalpha 0/0 and TRbeta -/- mice to produce double-heterozygous animals, 8 times. The double-heterozygous mice were mated to produced double-homozygous mice, which were backcrossed 3-5 times to each other.

Mice were weaned on the 4th wk after birth and were fed Purina Rodent Chow (0.8 ppm iodine) ad libitum and tap water. They were housed, <= 5 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 days old at the time they were killed. At various intervals, ~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 them a low-iodine (LoI) diet supplemented with 0.15% propylthiouracil (PTU; Harlan Teklad, Madison, WI). On the 10th day, one group of mice maintained on the LoI/PTU diet (>5 mice/genotype) was injected daily for 4 days with vehicle only (1× PBS, controls), and another group received 0.2 µg of 3,3',5-triiodo-L-thyronine (L-T3) · 25 g body wt-1 · day-1. Experiments were terminated by exsanguination 14-16 h after the final injection. L-T3 dissolved in PBS or 0.002% human serum albumin as a vehicle was given by intraperitoneal 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% 1× PBS containing 5 mM NaOH and kept at -20°C, protected from light. Concentration of L-T3 was confirmed by RIA (Diagnostic Products, Los Angeles, CA). Blood samples were obtained at baseline, on the 10th day after the initiation of the LoI/PTU diet, and at the termination of the experiment on day 15.

The dose of L-T3 given to TH-deficient animals was derived from previous experiments. It was optimized to achieve a partial suppression of serum TSH to make evident the differences between WT and SRC-1-/- mice (22, 34, 38). This allowed us to examine the effect of deficiency of receptor and coactivator under identical conditions of TH supply. Metabolism of T3 was determined in each genotype by measuring serum T3 levels at 2, 4, 8, and 16 h after injection of L-T3 (22).

TH and TSH Concentrations in Serum

Serum TSH was measured in 50 µl of serum by use of a sensitive, heterologous, disequilibrium double-antibody precipitation radioimmunoassay, as previously described (28). Samples containing >200 mU of TSH/l were 5- and 50-fold diluted with TSH-deficient mouse serum.

Serum thyroxine (T4) and total T3 concentrations were measured by a double-antibody precipitation RIA (Diagnostic Products) with 25 and 50 µl of serum, respectively. The sensitivities 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.

Serum Leptin

Samples were taken from frozen sera (obtained by retroorbital bleed and stored at -80°C), and leptin levels were determined by RIA (Linco Research, St. Charles, MO) with 10 µl of serum diluted into 100 µl in duplicate. Results are reported as serum leptin in nanograms per milliliter.

Measurements of Growth, Heart Rate, and Energy Expenditure

Mice from 2 to 9 wk of age were weighed on a 200-g balance on the 1st day of each week. In addition, the length of each mouse was determined from the tip of the nose to the base of the tail under light gas anesthesia at the time of weighing. Heart rates of mice were determined at baseline, after 14 days of a LoI/PTU diet, and on a PTU diet following 14-16 h after the fourth daily intraperitoneal injection of T3 (0.2 µg/25 g). Animals were anesthetized with chloral hydrate (4 mg/10 g body wt ip), and heart rate was determined using a Hewlett-Packard model 78534AA monitor/terminal with a chart speed of 25 mm/s. Body temperature was maintained by keeping mice on a heating pad during measurement. Energy expenditure (EE) was determined at baseline by measurement of change in body weight and food consumption over 4 days as previously described (5, 37). EE was calculated according to the formula
EE (kcal/day) 

= [food consumption (g/day) × 4.058* × 0.8**]

± [weight change (g/day) × 7***]
where * is the caloric value of the food (in kcal/g), ** is adjustment for 20% of the food wasted in each litter as determined by bomb calorimetry in euthyroid WT mice, and *** is the caloric value of 1 g body wt change. Loss of weight is added, and weight gain subtracted.

Isolation of Liver mRNA

Livers from animals were immediately frozen on dry ice and stored at -80°C. For RNA extraction, ~80 mg of liver tissue from individual mice were homogenized in 1 ml of TRIzol (Life Technologies, Rockville, MD) with a Polytron tissue homogenizer. 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 1/10 volumes of 3 M NaOAc and 3 volumes of 100% ethanol at -80°C.

Microarray Analysis of Mouse Liver

Six mice (male, 70 days old) of three genotypes (WT, TRbeta -/-, or SRC-1-/-) were treated with PTU and intraperitoneal L-T3, as described in Induction of Hypothyroidism and Treatment with TH. Mice were killed on the 15th day, and livers were removed, immediately frozen on dry ice, and stored at -80°C. Livers from three mice were pooled into one sample used to extract RNA and make cDNA. The cDNA was hybridized to Clontech's mouse Atlas Microarray V1.2 (1,172 genes) and resolved on a phosphorimager (Clontech, Palo Alto, CA). Each microarray compared expression in TH-deprived liver tissue with that in L-T3-treated mouse liver for each genotype. With use of threshold ratios for significance of 1.67 for each comparison (WT with and without L-T3, TRbeta -/- with and without T3, and SRC-1-/- with and without L-T3), genes were identified in each group that were either up- or downregulated in response to TH. In all three groups, 7.8-8.9% of the total 1,172 genes displayed a potential response to TH. In WT and SRC-1-/- mice, a majority of responsive genes were upregulated by TH, whereas in the TRbeta -/- mice, a majority of responsive genes were downregulated by TH (Table 1).

                              
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Table 1.   Genes up- and downregulated by TH in WT, TRbeta -/-, and SRC-1-/- mice

TaqMan RT-PCR of Genes Expressed in Liver

To quantitate mRNA expression of various genes in livers of different genotypes, 2 µg of total RNA were reverse transcribed using the First-Strand Synthesis Superscript Kit (Life Technologies) according to the provided protocol. Reverse transcription (RT) was performed using random hexamers. cDNAs obtained from the RT reaction were diluted with RNAse-free water to a concentration of 1 ng/µl. TaqMan fluorescent probe/primer sets were designed using Primer Express 1.5 (Applied Biosystems, Foster City, CA) and mRNA sequences taken from GenBank. Specificity was confirmed by the Basic Logical Alignment Search Tool search. Primer/probe sets were then obtained for osteopontin, glutathione S-transferase (GST), split hand-split foot (SHSF), Ets-related transactivation factor (ERF), and 5'-deiodinase (MegaBases, Evanston, IL) genes (Table 2). Equal loading of wells was controlled using a commercially available probe/primer set for 18S ribosomal RNA (Applied Biosystems). Detection of mRNA was performed with sequence detector software and the ABI 7000 Sequence Detection System (Applied Biosystems), which is capable of reading two fluorophores (sample probe and 18S ribosomal control probe) simultaneously in each well.

                              
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Table 2.   Probe/primer sets for TaqMan quantitative real-time PCR used for mRNA quantitation

Ten nanograms of each reverse-transcribed cDNA sample were run in duplicate, and the reaction was performed with TaqMan Universal Mix and 96-well optical plates (Applied Biosystems). Each duplicate sample represents reverse-transcribed total RNA from individual mouse liver samples. 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 threshold cycles, the first corresponding to amplification of 18S rRNA (VIC fluorophore), and the second to a specific gene of interest (FAM fluorophore). Normalization of data involved subtraction of rRNA Ct from that of the specific gene being amplified per well, because amplification is logarithmic. For each mouse genotype analyzed, at least five individual liver sample RNAs were run in duplicate. To calculate results, the average Ct for WT mice was determined. Individual mouse liver data were reported as degrees of increase or decrease from this WT average. Assays were repeated >= 3 times, and the data were normalized and merged.

TaqMan quantitative RT-PCR expression data for the four genes identified by microarray analysis (osteopontin, GST, SHSF, and ERF) are reported as percent change of PTU-treated mice from littermates on a PTU diet treated with T3 (as described in Induction of Hypothyroidism and Treatment with TH). The mean degree of change of PTU-treated animals of each genotype was determined relative to that of WT mice on PTU. Percent change was calculated by dividing each genotype's T3-treated mean (calculated against WT PTU) by its own PTU mean (±SE). Each group of animals (PTU and PTU+T3) had at least five mice.

Data Presentation and Statistics

Values are reported as means ± SE. Initially, a two-way or one-way ANOVA calculation was done to determine whether there was a significant interaction between treatment and genotype on each of the parameters measured. If significant interaction was detected, the effect of treatment was examined separately for each genotype, and vice versa. The Tukey-Kramer method, at 5% significance (Statview v. 5.0, SAS Institute), was used to control for multiple comparisons. To stabilize the variance of data for serum TSH and leptin, these data were analyzed on a logarithmic scale.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thyroid Function Tests

Thyroid function tests of different mouse genotypes at baseline and after treatment with PTU or PTU and intraperitoneal T3 are shown in Table 3. Experiments were performed in adult male mice because of previously reported sex and age differences in TH levels in WT mice (5, 28). Serum total T4 and T3 levels, as well as TSH concentrations, were significantly higher in TRbeta -/- and SRC-1-/- compared with WT mice, as previously reported (12, 34, 38). TRalpha 0/0 mice demonstrated decreased serum T4 with normal TSH compared with WT mice (P < 0.005). In the combined TRbeta -/-SRC-1-/- mice, serum levels of T4, T3, and TSH were 1.7, 2.7, and 2.8 times greater, respectively, than in TRbeta -/- mice or 2.7, 2.8, and 10 times greater, respectively, than in the SRC-1-/- mice. Mice deficient in both SRC-1 and TRalpha had serum T4 and T3 values that were 2.0 and 1.7 times greater than TRalpha 0/0, respectively, and TSH values were also increased by 2.3 times. Levels of free T3 and T4 showed similar differences (35).

                              
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Table 3.   Thyroid function tests

TH deprivation resulted in marked increases in serum TSH in all genotypes, although serum TSH levels in TRalpha 0/0 and TRalpha 0/0SRC-1-/- mice did not increase as much as in WT mice, and TSH levels in TRbeta -/-SRC-1-/- mice increased threefold more than in WT (P < 0.0001). T4 levels in all mice decreased to <0.25 µg/dl after the PTU/LoI diet. Treatment with TH resulted in variable suppression of absolute serum TSH values depending on the genotype of the mouse. The TRbeta -/-SRC-1-/- mice had the least suppression, indicative of the highest degree of resistance to TH, and TRalpha 0/0 mice showed the highest suppression of serum TSH; the latter did not reach statistical significance because of the large standard deviation of WT mice.

Growth in Mice of Different Genotypes

Length and body weight from age 2 to 9 wk were measured in mice of different genotypes (Fig. 1). SRC-1-/- mice grew at the same rate and achieved similar lengths at week 9 compared with WT mice. At week 5, lengths of TRbeta -/- and TRalpha 0/0 mice were 14% less than those of WT mice (significance at the 5% confidence level) and remained from 5 to 10% less compared with WT mice at week 9. Therefore, both TRalpha and TRbeta are required for normal linear growth. TRbeta is the major determinant of overall body length, because in the absence of TRbeta (with or without SRC-1), these animals have the most stunted length (Fig. 1A). TRalpha 0/0SRC-1-/- mice show more retarded linear growth than TRalpha 0/0 mice (Fig. 1B).


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Fig. 1.   Growth of mice of different genotypes from 2 wk until adulthood. Length of mice of each genotype was determined from age 2 wk to age 9 wk (top). Body weight was determined for the same time period of time (middle). Comparisons are shown for wild-type (WT), thyroid hormone receptor (TR)beta -/-, steroid receptor coactivator (SRC)-1-/-, and TRbeta -/-SRC-1-/- mice (left, A and C) and for WT, TRalpha 0/0, SRC-1-/-, and TRalpha 0/0SRC-1-/- mice (right, B and D). Nos. of animals in each group are shown in parentheses. Values in curves represent the mean ± SE of each group. Where no SE is present, values were sufficiently low as to not appear in graph. A table of significance is shown below graphs. Comparison was made between WT mice and other genotypes at 5 wk and 9 wk by the Tukey-Kramer method at 5% confidence.

Weight gain remained similar for TRbeta -/-, SRC-1-/-, and WT mice through week 9. For animals to achieve normal body weight, SRC-1 appears to be an important modifier in the absence of either TRalpha or TRbeta . TRalpha 0/0 mice have a 28% reduction in body weight at 3 wk and by 9 wk still maintain a 17% reduction in body weight (Fig. 1D). In the absence of both SRC-1 and TRalpha or TRbeta , mice have greater reduction in growth. Specifically, TRbeta -/-SRC-1-/- mice begin to have a decline in body weight at 8 wk (P < 0.0001), a change not seen in the SRC-1-/- or TRbeta -/- mice.

EE and Serum Leptin

EE experiments were performed in mice of 6-10 wk of age (Table 4). TRbeta -/-SRC-1-/- mice have the highest EE, 1.4 times that of WT mice (significance at the 5% confidence level). This, along with the high TH levels in these mice, implies that the TH-mediated EE occurs through TRalpha . This corresponds to the decrease in body weight seen in the TRbeta -/-SRC-1-/- mice (Fig. 1C). TRalpha 0/0 mice tend to have lower EE, but it does not reach significance. In the absence of SRC-1, TRbeta and/or TRalpha mice also maintain normal EE.

                              
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Table 4.   Baseline energy expenditure and leptin

Serum leptin concentrations in nonfasting mice were 2.1-fold higher in SRC-1-/- mice (P = 0.0472) and 2.5-fold higher in TRbeta -/-SRC-1-/- mice (P = 0.020) compared with WT mice (Table 4, Fig. 2). The higher leptin levels in these animals suggest that TH-mediated increase in leptin occurs in the absence of SRC-1 and TRbeta .


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Fig. 2.   Relationship of serum thyroxine (T4) levels and energy expenditure (EE; kcal · day-1 · g body wt-1; top) and nonfasting serum leptin (ng · ml-1 · g body wt-1; bottom). Values represent means. Note that EE and leptin and T4 levels were highest in the TRbeta -/-SRC-1-/- mice.

The contrasting effect of TH and genotype in EE and leptin concentration is demonstrated in Fig. 2. Note that EE did not increase very much despite significant increases in TH levels in the different genotypes, except for that in the combined TRbeta -/-SRC-1-/- mice. On the other hand, regulation of serum leptin levels is less dependent on genotype, but more dependent on TH levels, where the increase in TH is relatively independent of the genotype.

Heart Rate

Heart rates were measured in mice of different genotypes at baseline and after 14 days of a LoI/PTU diet or LoI/PTU with T3 treatment (Table 5). As previously reported at baseline (22), we found that TRalpha 0/0 mice had relative bradycardia compared with WT mice (371 ± 28 vs. 524 ± 10 beats/min, respectively; P < 0.0001). Unexpectedly, SRC-1-/- mice also had bradycardia (388 ± 12; P < 0.0001). Deletion of both TRalpha and SRC-1 resulted in the lowest mean heart rate. However, this value was not significantly different from values in the TRalpha 0/0 and SRC-1-/- mice just described. We also found baseline heart rates in TRbeta -/- mice to be lower than those in WT (P = 0.02). Mice showed decreases in heart rate in response to TH deprivation (LoI/PTU, Table 5), except for animals deficient in TRalpha . Although all genotypes increased heart rate in response to TH treatment, TRalpha 0/0SRC-1-/- mice had a modest increase of 17.6 ± 3.8% compared with 27-43% observed in the other genotypes. These data suggest that SRC-1 facilitates the TRalpha -mediated TH action in the heart.

                              
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Table 5.   Heart rates of mice at baseline and after TH withdrawal and TH treatment

Identification of TH-Responsive Genes by Microarray Analysis

TH-responsive genes specifically mediated by TRbeta or SRC-1 were identified by microarray analysis. Microarrays of mRNA from liver of WT, SRC-1-/-, and TRbeta -/- mice were analyzed, comparing each genotype in hypothyroid and TH-treated states. Analysis of over 1,100 genes revealed 92-105 genes that were affected by TH in each group (Table 1). Using high stringency (>2-fold change), we identified two genes that were TRbeta dependent (i.e., in which there was no effect of TH treatment in TRbeta -/- mice): osteopontin (upregulated in WT mice) and GST (downregulated in WT mice). Also, we identified two genes that were SRC-1 dependent (i.e., in which there was no effect of TH treatment in SRC-1-/- mice): SHSF (upregulated in WT mice) and ERF (downregulated in WT mice) (Table 6).

                              
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Table 6.   Genes identified to be up- or downregulated in response to TH and dependent on either TRbeta or SRC-1

Gene Expression in Liver with TH Withdrawal and TH Treatment

Confirmation of gene expression observed in the microarray analyses was done by TaqMan quantitative real-time PCR with the same and additional livers.

Osteopontin (J04806). WT and SRC-1-/- mice had a 76 and 30% increase, respectively, in osteopontin expression with T3 treatment. RNA from livers of TRbeta -/- and TRbeta -/-SRC-1-/- mice had no response to T3, confirming a TRbeta dependence for the TH-mediated upregulation of osteopontin demonstrated by microarray. Although not evaluated on the microarray, TRalpha also appears to be necessary for regulation of osteopontin expression, as TRalpha 0/0 and TRalpha 0/0SRC-1-/- mice failed to stimulate expression with TH treatment (Table 7). SRC-1 is not absolutely required for induction of osteopontin by TH, although it may facilitate the response, a 1.36 ± 0.16-fold (30.6 ± 0.16%) increase in SRC-1-/- compared with a 1.86 ± 0.39-fold increase (82.2 ± 50.7%) in WT.

                              
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Table 7.   Degree of induction with L-T3 treatment of various genes measured by TaqMan PCR

GST (J03958). GST expression appeared to be downregulated by TH on the basis of data from the microarray in WT mice, and it was also TRbeta dependent. Results from quantitative analysis are consistent with this data, showing an 0.21 ± 0.02-fold (81.7 ± 2.1%) decrease in GST expression in response to TH. In the absence of TRbeta , there was a small, paradoxical increase in GST expression in response to TH (Table 7). Interestingly, in the absence of both TRbeta and SRC-1, TH-mediated downregulation was restored (0.23 ± 0.04-fold; -77.8 ± 5.7%). This result indicates that the two receptor subtypes work together to facilitate downregulation of GST in response to TH. However, in the presence of only the TRalpha (TRbeta -/- mice), the additional presence of SRC-1 inhibits TH-mediated downregulation of GST. By eliminating the SRC-1 along with TRbeta , the inhibition of TH-mediated GST repression is removed.

SHSF (U41606). In WT mice, SHSF expression was increased by 1.5 ± 0.08-fold with TH treatment, and although a blunted response was seen in SRC-1-/- mice (1.19 ± 0.09), it was not significantly different from that in WT mice. The other genotypes also showed reduced responses (Table 7).

ERF (U58533). ERF was shown to be SRC-1 dependent by microarray, and we have confirmed this to be the case by quantitative PCR (Table 7). However, unlike the TH-mediated downregulation of ERF seen by microarray in WT and TRbeta -/- mice, quantitative RT- PCR results demonstrated a modest increase (1.59 ± 0.22 and 1.90 ± 0.38, respectively) in ERF expression. Furthermore, TRalpha 0/0 mice did not demonstrate any effect of TH.

5'Deiodinase (5'DI, NM007860). Although not included as part of the microarray, we chose to study 5'DI, a gene well known to be upregulated by TH. To evaluate the role of the TR subtypes and SRC-1 in mediating 5'DI expression, we investigated the response of 5'DI to TH in mice deficient in either TR subtype, in SRC-1, or in SRC-1 and each of the TR subtypes with TH deprivation and TH treatment (Fig. 3). There was a 300% increase in 5'DI expression in WT, SRC-1-/-, and TRalpha 0/0SRC-1-/- mice in response to TH. An even greater response (>2,400%) was observed in the TRalpha 0/0 mice, consistent with the hyperresponsiveness observed with other markers of TH action in these animals. This increased response was abrogated when SRC-1 was deleted along with the TRalpha . This result indicates that the hypersensitivity seen in the TRalpha 0/0 mice may be due, in part, to the presence of SRC-1, because in the absence of both, the increased response is ablated. TRbeta -/- mice had a markedly reduced response (only ~2.6% of the WT with TH), a reduction further compounded by deficiency in both TRbeta and SRC-1 (1.3% of WT with TH).


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Fig. 3.   mRNA quantitation of 5'-iodothyronine deiodinase (5'DI) in liver. 5'DI expression was determined by TaqMan quantitative real-time PCR. Data are shown as %increase (on a logarithmic scale) from propylthiouracil (PTU)-treated WT mice when also given injections of 3,3',5-triiodo-L-thyronine (T3) for 4 days (0.2 µg · 25 g-1 · day-1). The %increase is determined from mean liver expression of these genes from >= 5 mice in each treatment group and each genotype. SE are determined from % of change in individual samples from PTU-treated WT mice. Five mice were used for each hypothyroid and T3-treated group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies investigate the in vivo effect of TR subtypes in peripheral tissues, specifically by focusing on animal growth, heart rates, EE, and markers of TH action in the liver and heart. To do this, we have bred mice deficient in the TRalpha or TRbeta , in SRC-1, or in each of the TR subtypes and SRC-1 together. These animals were generated in a mixed sv129 and C57BL/6J background. Therefore, single- and double-heterozygous mice were backcrossed with WT littermates to generate a uniform genetic background closer to C57BL/6J. The role of the TR subtypes in growth has been studied previously in TR-deficient animals by our laboratory and others (10-12, 16, 30). In addition, mice deficient in SRC-1 have no reported changes in linear growth and weight (42). Here, we show that, at 5 wk, mice deficient in TRbeta or SRC-1 have body weights similar to those of WT mice. However, we show somewhat decreased linear growth in TRbeta -/- mice relative to WT mice at 5 wk (86.8%, significance at the 5% confidence level) and linear growth at 9 wk (90.9%, significance at the 5% confidence level). Although we and others have previously reported no differences in body weight and linear growth in TRbeta -/- mice, the trends are there, and with our larger number of mice in this study, differences reach significance. These changes are much milder than in mice deficient in TRalpha at 5 wk (74.6%; 86.8%), a result consistent with work showing that TRalpha 1 is important for intestinal crypt development at weaning, a phenotype that seems to improve (27). At 9 wk, TRalpha 0/0 mice still show decreased linear and body weight growth (86.8%; 95.0%), similar to what has been shown previously (12, 13). However, after full maturity, both TRalpha - and TRbeta -deficient mice have retarded growth. Although SRC-1-/- mice achieve adult linear and body weights that are not different from those of WT animals, loss of SRC-1 in combination with either TR subtype (TRalpha 0/0SRC-1-/- or TRbeta -/-SRC-1-/-) results in linear and weight growth retardation compared with WT at both 5 wk (86.7/92.2% and 69/76.9%, respectively) and 9 wk (77.7/73.0% and 90.8/90.0%, respectively). Taken together, these data indicate that both TR subtypes have important roles in governing linear and body weight growth. In addition, SRC-1 plays a cooperative role in growth with each TR subtype in the absence of the other. In the absence of SRC-1, the presence of other cofactors may compensate for its loss, but insufficiently when there is additional loss of either TR. Although we did not investigate expression of growth hormone in TR subtype/SRC-1-deficient mice, we speculate that these levels may be normal, as has been shown in TRalpha 0/0 (13), TRalpha 1-/- and TRbeta -/- (13), and SRC-1-/- (32) mice. Additionally, animals deficient in both TRbeta and SRC-1 show increased EE over all other genotypes on a normal diet (P < 0.001). This corresponds with much higher baseline levels of TH (Table 3) and progressive weight loss in these mice at 9 wk. The use of a TRbeta isoform-specific ligand, GC-1, failed to maintain core body temperature and reduced stimulation of uncoupling protein in brown adipose tissue of hypothyroid mice (29). Taken together, these data indicate that TRalpha mediates the increased EE observed in response to TH, supporting previous observations (39), and that SRC-1 is not necessary for this action of TH. The increased EE and weight loss in these animals do not correspond to illness, as these animals demonstrate fertility and litter size at homozygosity that are similar to those of WT, without increased mortality in adult animals, at least through 60 wk, the longest we have maintained them.

That TRalpha is required for normal heart rate has been shown previously (15, 19, 20, 22, 30). In addition, it has also been reported by our laboratory and others that absence of TRbeta does not cause such a decline in basal heart rates and, in fact, results in a slight increase in heart rate attributed to increased circulating levels of TH in these mice (Table 3) (15, 19, 20, 33, 35, 36). However, there appear to be a number of differences in baseline heart rates of WT mice dependent on strain, even in papers by the same laboratories. SRC-1 appears to be essential for maintaining heart rate, as in its absence, alone and in combination with each of the receptor subtypes, heart rate is decreased. In the absence of SRC-1, TRalpha and TRbeta are unable to maintain normal heart rates, presumably by competing for a limited supply of cofactors or an affinity of the TRalpha 2, which does not bind TH, for other cofactors. In the absence of TRbeta alone, TRalpha and SRC-1 are unable to maintain WT heart rate. However, in the absence of both TRbeta and SRC-1, there is a marked increase in serum TH (Table 3) over that in TRbeta -/- mice available to bind to TRalpha 1, which does not happen in the absence of SRC-1 alone. This seems likely, in that TRbeta -/-SRC-1-/- mice achieve WT heart rates when treated with exogenous TH. Despite the proposed role for SRC-1 in regulating heart rate that is inferred by these data, we have previously shown that other TH-dependent genetic markers in heart, specifically SERCA2, MHCalpha , and MHCbeta , are unaffected by the absence of SRC-1 (32).

To discern the mechanism of TH action in the liver, we sought to identify genetic markers that were TRbeta dependent vs. SRC-1 dependent. It has been shown in vitro that coactivators are used by TRbeta to mediate TH action (reviewed in Ref. 23). We took livers from WT, TRbeta -/-, and SRC-1-/- mice that were made hypothyroid with a LoI/PTU diet or were hypothyroid and treated with TH, and we subjected them to microarray analysis. From this analysis, we identified four TH-dependent genes; two were determined to be TRbeta dependent (osteopontin, upregulated, and GST, downregulated) and two were SRC-1 dependent (SHSF, upregulated, and ERF, downregulated). Osteopontin is a secreted glycoprotein with an RGD domain characteristic of integrin-binding proteins. It has been shown to be an important chemokine in inflammation, a potential oncogene in renal cancers, a stable component in mineralized tissues (interacting with the vitamin D receptor and the retinoid X receptor) and smooth muscle, with an additional presence in the anterior pituitary (6, 26). A direct role for osteopontin regulation by TH has not been shown. Data here confirmed microarray results showing that osteopontin expression was indeed TRbeta dependent, as mice deficient in TRbeta (TRbeta -/- and TRbeta -/-SRC-1-/-) showed no response to TH (Fig. 3A). We also showed that osteopontin may also be regulated by the TRalpha , as mice deficient in TRalpha (TRalpha 0/0 and TRalpha 0/0SRC-1-/-) show a decrease in osteopontin in response to TH (-16.9 and -26.3%, respectively), distinguishing the roles for TR subtypes in controlling this gene. Flores-Morales et al. (9) demonstrated that 40% of TH-responsive genes identified in an expression profile were TRbeta independent, also suggesting a role for TRalpha in modulating gene expression. In addition, we investigated GST by RT-PCR. Beckett and colleagues (3, 4) showed that, by depriving rats of selenium in their diets (inhibiting T3 production in liver by 5'DI) or by use of the PTU diet, there was in increase in expression of GST. However, there has been no detailed study on the effects of administration of exogenous L-T3 on GST expression. Here, we show in TRbeta -/- mice that there is no downregulation of GST as seen in other genotypes, which suppress GST with TH treatment by ~75% (Table 7). Interestingly, in the absence of TRbeta and SRC-1, suppression by TH absent in TRbeta -/- mice is restored. From this, we conclude that, in TRbeta -/- mice, the presence of SRC-1 inhibits TRalpha -mediated suppression of GST, and when SRC-1 is also removed, the TRalpha , possibly acting with another coregulatory molecule, mediates TH-induced GST suppression.

Of the two TH-responsive genes identified to be SRC-1 dependent, we were unable to confirm the microarray data with RT-PCR. In WT mice, SHSF increased by 40% without any response in SRC-1-/- mice (Table 7). However, there were also blunted responses to TH in the other genotypes investigated, indicating that SHSF will not be a good marker for further use. ERF was only partially SRC-1 dependent by RT-PCR; however, we saw differences between TaqMan and microarray in the response of WT and TRbeta -/- mice to TH, showing increases in ERF expression (Table 7) vs. decreases seen by microarray (Table 6). It is possible that ERF would be a good marker for future use, but it would require further investigation. Importantly, we confirmed the viability of two new TRbeta -dependent markers (osteopontin and GST) in the presence of TH. These genes have not been previously identified in two other studies of TH-responsive gene expression (8, 9). We have seen other genes reported by microarray that were not reproduced by other methods (Weiss RE and Sadow PM, unpublished data) and note that the previous studies confirmed only a small number of the genes with Northern analysis that were identified by microarray (8, 9).

In addition to studying new markers identified by microarray, we also investigated by RT-PCR liver expression of 5'DI, an enzyme whose expression has been known to be upregulated by TH via the TRbeta (1). Interestingly, we saw sharp increases in 5'DI expression in TRalpha 0/0 mice, far greater than in any other genotype (Fig. 3). Macchia et al. (22) recently reported that mice deficient in all known TRalpha isoforms have hypersensitivity to TH. It was speculated that a potential mechanism for hypersensitivity in these animals would be elimination of the inhibitory TRalpha 2. However, as 5'DI data would indicate, the hypersensitivity conferred upon TRalpha -deficient mice (evidenced by vast TH-induced increases in 5'DI expression) is ablated in the absence of both TRalpha and SRC-1, in which TRalpha 0/0SRC-1-/- mice increase 5'DI in response to TH to levels similar to those of WT and SRC-1-/- animals. This result indicates that the hypersensitivity seen in TRalpha 0/0 mice may be due to more than absence of the inhibitory TRalpha 2. In fact, it is likely that the hypersensitivity is due to an increased availability of SRC-1 to interact with the TRbeta , an event that may be controlled in TR-competent mice by squelching of the coactivator by the TRalpha .

A model of TH action in liver is shown in Fig. 4. 5'DI represents a liver gene upregulated by TH. TRbeta 1 appears to be the key isoform to mediate TH action on 5'DI in the liver, because in its absence, there is a major reduction in 5'DI induction with TH. In TRalpha 0/0 mice, there is a hyperresponse in 5'DI to TH. We propose that this TH hypersensitivity is due to relief of inhibition by TRalpha 2, as originally hypothesized by Macchia et al. (22). However, we extend this hypothesis to include that SRC-1 is necessary for this action in liver and that SRC-1 inhibits TRalpha 2 activity, as TRalpha 0/0SRC-1-/- mice are not hypersensitive to TH-induced 5'DI expression. This result might best be confirmed by overexpression of SRC-1 in vivo in liver and observation of the effect on gene expression. Additionally, we propose that SRC-1 facilitates TRbeta -induced 5'DI expression but is not necessary. It is possible that, in the absence of SRC-1, alternative coactivators, such as TIF-2 or SRC-3, could compensate.


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Fig. 4.   Model for thyroid hormone action in the liver. Arrows represent activation, and blockers represent inhibition. Thickness of lines or no. of arrows represents potential strength of interaction.

From these studies, we conclude that 1) in the absence of TRalpha or TRbeta , SRC-1 is important for normal growth; 2) SRC-1 partially mediates the TH effect on heart rate by TRalpha and TRbeta ; 3) we have identified two new TH-responsive markers in the liver mediated by TRbeta : osteopontin (upregulated) and glutathione S-transferase (downregulated); and 4) we have shown that SRC-1 may mediate the hypersensitivity to TH seen in TRalpha 0/0 mice, as demonstrated by 5'deoiodinase expression in liver.


    ACKNOWLEDGEMENTS

We are indebted to Prof. Samuel Refetoff for advice and guidance during this project and to Dr. T. Karrision for help with statistical analyses. We also gratefully acknowledge the technical assistance of Kevin Cua with preliminary energy expenditure and heart rate experiments.


    FOOTNOTES

This work was supported in part by grants from the National Institutes of Health: DK-58281 (to R. E. Weiss), DK-58242 (to J. Xu), and HD-078587 (to B. W. O'Malley), and from the Ministry of Research ACI 283 (to J. Samarut), and by the Seymour J. Abrams Thyroid Research Center.

Address for reprint requests and other correspondence: R. E. Weiss, Thyroid Study Unit, MC 3090, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: rweiss{at}medicine.bsd.uchicago.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

September 17, 2002;10.1152/ajpendo.00226.2002

Received 23 May 2002; accepted in final form 7 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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