Specificity of Thyroid Hormone Receptor Subtype and Steroid Receptor Coactivator (SRC)-1 on Thyroid Hormone Action

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Specificity of Thyroid Hormone Receptor Subtype and Steroid Receptor

Coactivator (SRC)-1 on Thyroid Hormone Action

Peter M. Sadow1, 2, Olivier Chassande3, Karine Gauthier3, Jacques Samarut3, Jianming Xu4, Bert

W. O’Malley4, and Roy E. Weiss1,5

1Depts. of Medicine and 2Pathology, The University of Chicago, Chicago, IL 60637;

3Laboratoire de Biologie Moléculaire et Cellulaire de l’Ecole Normale Supérieure de Lyon,

Lyon, France; 4Department of Molecular and Cellular Biology, Baylor College of Medicine,

Houston, TX 77030.

5Corresponding Author:

Roy E. Weiss, MD, PhDThyroid Study Unit, MC 3090University of Chicago5841 S. Maryland AveChicago, IL USAEmail: [email protected]: 773-702-9266Fax: 773-834-3966

Running Title: SRC-1 and TR Specificity in peripheral tissues

Copyright 2002 by the American Physiological Society.

AJP-Endo Articles in PresS. Published on September 17, 2002 as DOI 10.1152/ajpendo.00226.2002

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Abstract

Isoforms of the thyroid hormone receptor (TR) and TR 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 modulates 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 TRα and SRC-1 (TRα0/0SRC-1-/-), as well as in TRβ and SRC-1 (TRβ-/-SRC-1-/-) and

investigated thyroid function tests and effects of TH deprivation and TH treatment compared to

wild type (WT) mice or those deficient in either TR or SRC-1 alone. The data show that (1) in

the absence of TRα or TRβ, SRC-1 is important for normal growth; (2) SRC-1 modulates TRα

and TRβ effects on heart rate; (3) two new TRβ-dependent markers of TH action in the liver

have been identified, osteopontin (up-regulated) and glutathione-S-transferase (down-regulated);

and (4) SRC-1 may mediate the hypersensitivity to TH seen in liver of TRα0/0 mice.

Keywords: knockout/ resistance to thyroid hormone/ SRC-1/ thyrotropin/ thyroid hormone

receptor

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Introduction

Thyroid hormone receptor (TR) α and TRβ function as nuclear transcription factors that

mediate thyroid hormone (TH) action. TRs bind to TH response elements (TREs) 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

(26).

Since some genes are up-regulated and others are down-regulated by TH in same cell,

various theories have been proposed to explain the mechanism(s) of TR- modulated gene

expression (see (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 TRβ knockout mice

(TRβ-/-) have resistance to thyroid hormone (RTH) (10-12, 22, 34, 37), while mice with

disruption of the TRα1 and 2 isoforms (TRα0/0) 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

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completely deficient in both TRβ and TRα exhibit more severe resistance to TH than those

lacking TRβ 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 (37, 42). Therefore we ask whether SRC-1 differentially modulates the functions of

TRα and TRβ, and if so, how does this effect influence TH action in the liver and heart?

For this purpose we generated mice deficient in TRα and SRC-1 (TRα0/0SRC-1-/-), as

well as mice deficient in the TRβ and SRC-1 (TRβ-/-SRC-1-/-) and compared these to wildtype

(WT) mice or mice with deficiency in TRβ, TRα and SRC-1 alone. Our data provide evidence

that in the absence of TRα or TRβ, SRC-1 is important for normal growth. We also show that

SRC-1 mediates TRα and TRβ action in the heart. This study identifies two novel TRβ-

dependent markers of TH action in the liver, osteopontin (up-regulated) and glutathione-S-

transferase (down-regulated). We have also demonstrated that SRC-1 may mediate the

hypersensitivity to TH seen in TRα0/0 mice.

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Materials and Methods

Generation and handling of animals

Mice deficient in SRC-1 (SRC-1-/-) 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 Met-381 position and deleting 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, 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).

TRβ-/- mice were produced as previously described (12). These 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 TRβ1 and TRβ2 (12). The TRα0/0 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 TRα1 and

TRα2 transcripts, but also that of TR∆α1 or TR∆α2 by removing a transcription start site in

intron 7 (13). The gene sequence for rev-erbA α protein encoded by the opposite strands for the

TRα (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 at 3-9 times into the same strain, diluting the

129sv background.

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The individual knockout mice were backcrossed more than nine times on a C57BL/6

background to produce a uniform genetic background for wildtype and knockout animals. We

then crossed the SRC-1-/- mice with TRα0/0 and TRβ-/- 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 week after birth and were fed Purina Rodent Chow (0.8

ppm Iodine) 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 days old at the time of sacrifice. 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 following

centrifugation and stored at minus 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 (PTU, Harlan Teklad, Madison, WI). On the 10th

day, one group of mice (>5 mice/genotype) was injected daily for 4 days with vehicle only (1X

PBS, controls) while another received 0.2 µg of L-T3/25g body wt/day, maintained on the

LoI/PTU diet. Fourteen to 16 h after the final injection, experiments were terminated by

exsanguination. L-T3, dissolved in phosphate buffered saline and 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% 1X 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

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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 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 wildtype 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 following injection of L-T3 (22).

TH and TSH concentrations in serum

Serum TSH was measured in 50 µl of serum using a sensitive, heterologous,

disequilibrium double antibody precipitation radioimmunoassay as previously described (28).

Samples containing more than 200 mU TSH/L were 5- and 50-fold diluted with TSH-deficient

mouse serum.

Serum T4 and total T3 concentrations were measured by a double antibody precipitation

RIA (Diagnostic Products, Los Angeles, CA) using 25 and 50 µl of serum, respectively. The

sensitivities of these assays were 0.2 µg T4/dL and 5 ng T3/dL. The inter-assay 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 ng/mL.

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Measurements of Growth, Heart Rate and Energy Expenditure

Mice were weighed on 200 gm balance on the first day of each week from age 2-9 weeks.

In addition, 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, following 14 days of a LoI/PTU diet, and on a PTU diet following 14-16 hours after the

fourth daily IP injection of T3 (0.2µg/25g). Animals were anesthetized with chloral hydrate (4

mg/10 g body weight, ip) and heart rate was determined using a Hewlett Packard

Monitor/Terminal Model 78534AA with a chart speed of 25 mm/second. 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) x 4.058* x 0.8**) ± (weight change (g/day) x 7***)

Above, * is the caloric value of the food (in Kcal/g); ** is adjustment for 20% food

wasted in litter as determined by bomb calorimetry in euthyroid wildtype mice; *** is caloric

value of 1 g body weight 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, approximately 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 volume 3M NaOAc and 3

volumes of 100% ethanol at -80˚C.

Microarray Analysis of Mouse Liver

Six mice (male, age 70 days) of three genotypes (WT, TRβ-/- or SRC-1-/-) were treated

with PTU and ip L-T3 as described above. Mice were killed on the 15th day and livers were

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removed and immediately frozen on dry ice (and stored at -80˚C). Livers from 3 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

performed by Clontech (Palo Alto, CA). Each microarray compared expression in TH-deprived

liver tissue to that in L-T3-treated mouse liver for each genotype. Using threshold ratios for

significance of 1.67 for each comparison (WT with and without L-T3, TRβ-/- with and without T3,

and SRC-1-/- with and without L-T3), genes were identified in each group that were either up- or

down-regulated in response to TH. In all 3 groups, 7.8-8.9% of the total 1,172 genes displayed a

potential response to TH. In WT and SRC-1-/- mice, the majority of responsive genes were up-

regulated by TH, while in the TRβ-/- mice, the majority of responsive genes were down-regulated

by TH (Table I).

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TaqMan RT PCR of Genes Expressed in Liver

In order 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, Rockville, MD) 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 BLAST 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)

(Table II). Equal loading of wells was controlled using a commercially available probe/primer

set for 18S ribosomal RNA (Applied Biosystems, Foster City, CA). Detection of mRNA was

performed with Sequence Detector Software and the ABI 7000 Sequence Detection System

(Applied Biosystems, Foster City, CA), capable of reading two fluorophores simultaneously in

each well (sample probe and 18S ribosomal control probe).

10 ng 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,

Foster City, CA). 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 specific

gene of interest (FAM fluorophore). Normalization of data involved subtraction of rRNA Ct

from that of the specific gene being amplified per well, as 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 wildtype mice was determined. Individual mouse liver data

was reported as fold increase or decrease from this wildtype average. Assays were repeated at

least 3 times and the data were normalized and merged.

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TaqMan quantitative RT PCR expression data for the four genes identified by microarray

analysis (osteopontin, glutathione S-transferase (GST), split hand split foot (SHSF), and Est-

related factor(ERF)) is reported as % change of PTU-treated mice from littermates on a PTU diet

treated with T3 (as described above). The mean fold change of PTU-treated animals of each

genotype was determined relative to wildtype 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 mean ± SE. Initially a two-way or one-way ANOVA calculation

was done to determine if 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 Inc.) was used to control for multiple comparisons.

To stabilize the variance of data for serum TSH and Leptin, these data only were analyzed on a

logarithmic scale.

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Results

Thyroid function tests

Thyroid function tests of different mouse genotypes at baseline, and following treatment with

PTU or PTU and ipT3 are shown in Table III. 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 TRβ-/- and SRC-1-/- as

compared to WT mice as previously reported (12, 34, 38). TRα0/0 mice demonstrated decreased

serum T4 with normal TSH compared to WT mice (p<0.005). In the combined TRβ-/-SRC-1-/- mice,

serum levels of T4, T3 and TSH were 1.7, 2.7 and 2.8 times greater, respectively, than in TRβ-/- or

2.7, 2.8 and 10 times greater, respectively, than in the SRC-1-/- mice. Mice deficient in both SRC-1

and TRα have serum T4 and T3 values that were 2.0 and 1.7 times greater than TRα0/0, respectively,

and TSH values were also increased by 2.3 times. Levels of free T3 and T4 showed similar

differences (35).

TH deprivation resulted in marked increases in serum TSH in all genotypes, although serum

TSH levels in TRα0/0 and TRα0/0SRC-1-/- mice did not increase as much as in WT, and TSH levels in

TRβ-/-SRC-1-/- mice increased 3-fold greater than WT (p<0.0001). T4 levels in all mice decreased to

<0.25 µg/dl following PTU/LoI diet. Treatment with TH resulted in variable suppression of absolute

serum TSH values depending on the genotype of the mouse. The TRβ-/-SRC-1-/- mice had the least

suppression, indicative of its highest degree of resistance to TH and TRα0/0 mice showed the highest

suppression of serum TSH; the latter did not reach statistical significance due to the large standard

deviation of WT mice.

Growth in mice of different genotypes

Length and body weight from age 2 to 9 weeks 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 to

WT mice. At week 5, lengths of TRβ-/- and TRα0/0 mice were 14% less than WT mice (significance

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at the 5% confidence level) and remained from 5-10% smaller compared to WT mice at week 9.

Therefore, both TRα and TRβ are required for normal linear growth. TRβ is the major determinant

of overall body length, as in the absence of TRβ (with or without SRC-1) these animals have the

most stunted length (Fig. 1A). TRα0/0SRC-1-/- show more retarded linear growth than TRα0/0 mice

(Fig. 1B).

Weight gain remained similar for TRβ-/-, 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

the TRα or TRβ. TRα0/0 mice have a 28% reduction in body weight at 3 weeks and by 9 weeks still

maintain a 17% reduction in body weight (Fig. 1D). In the absence of both SRC-1 and either the

TRα or TRβ, mice have greater reduction in growth. Specifically, TRβ-/-SRC-1-/- mice begin to have

a decline in body weight at 8 weeks (p<0.0001), a change not seen in the SRC-1-/- or TRβ-/- mice.

Energy Expenditure and Serum Leptin

Energy expenditure experiments were performed in mice 6-10 weeks of age (Table IV).

TRβ-/-SRC-1-/- mice have the highest energy expenditure, 1.4 times that of WT mice (significance at

the 5% confidence level). This, along with the high TH levels in these mice, imply the TH-

mediated energy expenditure occurs through TRα. This corresponds to the decrease in body weight

seen in the TRβ-/-SRC-1-/- mice (Fig. 1C). TRα0/0 tend to have lower energy expenditure, but it does

not reach significance. In the absence of SRC-1, TRβ and/or TRα also maintain normal energy

expenditure.

Serum leptin concentrations in nonfasting mice were 2.1 fold higher in SRC-1-/- mice

(p=.0472) and 2.5 fold higher in TRβ-/-SRC-1-/- mice (p=0.020) compared to WT (Table IV, Fig. 2).

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The higher leptin levels in these animals suggest that TH mediated increase in leptin occurs in the

absence of SRC-1 and TRβ.

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 the combined TRβ-/-SRC-1-/- mice. On the other hand, regulation of

serum leptin levels are 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 V). As previously reported at baseline (22), we

find that TRα0/0 mice have relative bradycardia compared to WT mice (371 ± 28 beats/min versus

524 ± 10 beats/min, respectively; p<0.0001). Unexpectedly, SRC-1-/- mice also had bradycardia

(388 ± 12; p<0.0001). Deletion of both TRα and SRC-1 resulted in the lowest mean heart rate.

However, this value was not significantly different from those in the TRα0/0 and SRC-1-/- above.

We also found baseline heart rates in TRβ-/- mice to be lower than WT (p=0.02). Mice showed

decreases in heart rate in response to TH deprivation (LoI/PTU, Table V), except for animals

deficient in TRα. Although all genotypes increased heart rate in response to TH treatment,

TRα0/0SRC-1-/- mice had a modest increase of 17.6 ± 3.8% compared to 27-43% observed in the

other genotypes. These data suggest that SRC-1 facilitates the TRα- mediated TH action in the

heart.

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Identification of TH-responsive genes by microarray analysis

TH responsive genes specifically mediated by TRβ or SRC-1 were identified by

microarray analysis. Microarrays of mRNA from liver of wildtype, SRC-1-/-, and TRβ-/- 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 I). Using high

stringency (>2-fold change), we identified two genes that were TRβ-dependent (i.e., in which

there was no effect of TH treatment in TRβ-/- mice): osteopontin (up-regulated in WT mice) and

gluathione-S-transferase (GST) (down-regulated 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):

split hand split foot (SHSF) (up-regulated in WT mice) and Ets-related transactivation factor

(ERF) (down-regulated in WT mice) (Table VI).

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 in osteopontin expression with T3

treatment, respectively. RNA from livers of TRβ-/- and TRβ-/-SRC-1-/- mice had no response to

T3, confirming a TRβ-dependence for TH-mediated upregulation of osteopontin demonstrated by

microarray. Although not evaluated on the microarray, TRα also appears to be necessary for

regulations of osteopontin expression, as TRα0/0 and TRα0/0SRC-1-/- mice failed to stimulate

expression with TH treatment (Table VII). SRC-1 is not absolutely required for induction of

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osteopontin by TH, though it may facilitate the response, 1.36 ± 0.16 fold (30.6 ± 0.16%)

increase in SRC-1-/- compared to 1.86 ± 0.39 fold increase (82.2 ± 50.7%) in WT.

Glutathione S-transferase (J03958)

GST expression appeared to be down-regulated by TH based on data from the microarray

in WT mice and was also TRβ-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 TRβ, there was a small, paradoxical increase in GST expression in

response to TH (Table VII). Interestingly, in the absence of both TRβ and SRC-1, TH mediated

down-regulation is restored (0.23 ± 0.04 fold; -77.8 ± 5.7%). This result indicates that the two

receptor subtypes work together to facilitate down-regulation of GST in response to thyroid

hormone. However, in the presence of only the TRα (TRβ-/- mice), the additional presence of

SRC-1 inhibits TH-mediated down-regulation of GST. By eliminating the SRC-1 along with

TRβ, the inhibition of TH-mediated GST repression is removed.

Split Hand Split Foot (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 WT mice. The other genotypes also showed reduced responses (Table VII).

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Ets Related Factor (U58533)

ERF was shown to be SRC-1-dependent by microarray, and we have confirmed this to be

the case by quantitative PCR (Table VII). However, unlike the TH-mediated down-regulation of

ERF seen by microarray in WT and TRβ-/- mice, quantitative RT PCR results demonstrate a

modest increase (1.59 ± 0.22 and 1.90 ± 0.38, respectively) in ERF expression. Furthermore,

TRα0/0 mice did not demonstration any effect of TH.

5’Deiodinase (5’ DI, NM_007860)

Although not included as part of the microarray, we chose to study 5’DI, a gene well

known to be up-regulated by TH. In order 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, SRC-1, or SRC-1 and each of the TR subtypes with TH deprivation and TH

treatment (Fig. 3). There was a 300% fold increase in 5’DI expression in WT, SRC-1-/-, and

TRα0/0SRC-1-/- mice in response to TH. An even greater response (>2,400%) was observed in

the TRα0/0 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 TRα. This result indicates that the hypersensitivity seen in the TRα0/0 mice may be, in

part, due to the presence of SRC-1, as in the absence of both, the increased response is ablated.

TRβ-/- mice had a markedly reduced response (only about 2.6% of the wildtype with TH), a

reduction further compounded by deficiency in both TRβ and SRC-1 (1.3% of WT with TH).

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Discussion

These studies investigate the in vivo effect of TR subtypes in peripheral tissues,

specifically by focusing on animal growth, heart rates, energy expenditure and markers of TH

action in the liver and heart. To do this, we have bred mice deficient in the TRα or TRβ, SRC-1,

or 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 wildtype 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 weeks, mice deficient in TRβ

or SRC-1 have body weights similar to WT mice. However, we show somewhat decreased

linear growth in TRβ-/- mice relative to WT at 5 weeks (86.8%, significance at the 5% confidence

level) and linear growth at 9 weeks (90.9%, significance at the 5% confidence level). Although

we and others have previously reported no differences in body weight and linear growth in TRβ-/-

mice, the trends were there and with our larger number of mice, differences reach significance.

These changes are much milder than in mice deficient in TRα at 5 weeks (74.6%; 86.8%), which

is consistent with work showing that TRα1 is important for intestinal crypt development at

weaning, a phenotype that seems to improve (27). At 9 weeks, TRα0/0 mice still show decreased

linear and body weight growth (86.8%; 95.0%), similar to what has been shown previously

(12,13). However, following full maturity, both TRα- and TRβ- deficient mice have retarded

growth. Although SRC-1-/- mice achieve adult linear and body weights that are no different than

wildtype animals, loss of SRC-1 in combination with either TR subtype (TRα0/0SRC-1-/-/TRβ-/-

SRC-1-/-) results in linear and weight growth retardation compared to WT both at 5 weeks

E-00226-2002.R2

19

(86.7/92.2% and 69/76.9%, respectively) and at 9 weeks (77.7/73.0% and 90.8/90.0%,

respectively). Taken together, these data indicate that both TR subtypes play 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 TRα0/0 (13),

TRα1-/- and TRβ-/- (13) and SRC-1-/- (32) mice. Additionally, animals deficient in both TRβ and

SRC-1 show increased energy expenditure over all other genotypes on a normal diet (p<0.001).

This corresponds with much higher baseline levels of thyroid hormone (Table III) and

progressive weight loss in these mice at 9 weeks. The use of a TRβ 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 TRα

mediates the increased energy expenditure observed in response to TH supporting previous

observations (39) and that SRC-1 is not necessary for this action of TH. The increased energy

expenditure and weight loss in these animals does not correspond with illness, as these animals

demonstrate fertility and litter size at homozygosity that is similar to WT, without increased

mortality in adult animals, at least through 60 weeks, the longest we have maintained them.

That TRα 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 TRβ 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 III) (15, 19, 20, 33, 35, 36).

However, there appear to be a number of differences in baseline heart rates of WT mice

E-00226-2002.R2

20

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, TRα and TRβ are unable to maintain

normal heart rates, presumably by competing for a limited supply of cofactors or an affinity of

the TRα2, which does not bind TH, for other cofactors. In the absence of TRβ alone, TRα and

SRC-1 are unable to maintain WT heart rate. However, in the absence of both TRβ and SRC-1,

there is a marked increase in serum TH (Table III) over that in TRβ-/- mice available to bind to

TRα1, which does not happen in the absence of SRC-1 alone. This seems likely in that TRβ-/-

SRC-1-/- mice achieve WT heart rates when treated with exogenous TH. Despite the proposed

role for SRC-1 in regulating heart rate inferred by these data, we have previously shown that

other TH-dependent genetic markers in heart, specifically SERCA2, MHCα, and MHCβ, are

unaffected by the absence of SRC-1 (32).

In order to discern the mechanism of TH action in the liver, we sought to identify genetic

markers that were TRβ-dependent vs. SRC-1-dependent. It has been shown in vitro that

coactivators are used by TRβ to mediate TH action (reviewed in (23)). We took livers from WT,

TRβ-/- and SRC-1-/- mice that were made hypothyroid with a LoI/PTU diet or hypothyroid or

treated with TH and subjected them to microarray analysis. From this analysis, we identified

four TH-dependent genes; two were determined to be TRβ-dependent (osteopontin; up-

regulated) and glutathione-S-transferase (GST; down-regulated) and two were SRC-1-dependent

(split hand/split foot, SHSF; up-regulated) and Ets-related transactivation factor (ERF, down-

regulated). 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

E-00226-2002.R2

21

Vitamin D Receptor and the retinoid X receptor) and smooth muscle, with an additional presence

in the anterior pituitary (6, 25). A direct role for osteopontin regulation by TH has not been

shown. Data here confirmed microarray results that showed that osteopontin expression was

indeed TRβ-dependent, as mice deficient in TRβ (TRβ-/- and TRβ-/-SRC-1-/-) showed no response

to TH (Fig. 3A). We also showed that osteopontin may also be regulated by the TRα, as mice

deficient in TRα (TRα0/0 and TRα0/0SRC-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 of 40% of TH responsive genes identified in an

expression profile were TRβ independent, also suggesting a role for TRα in modulating gene

expression. In addition, we investigated GST by RT PCR. Beckett et al showed that by

depriving rats of selenium in their diets (inhibiting T3 production in liver by 5’DI) or by PTU

diet, there was in increase in expression of GST (3, 4). However, there has been no detailed

study on the effects of administration of exogenous L-T3 on GST expression. Here, we show in

TRβ-/- mice, there is no down-regulation of GST as seen in other genotypes, which suppress GST

with TH treatment by about 75% (Table VII). Interestingly, in the absence of TRβ and SRC-1,

suppression by TH absent in TRβ-/- mice is restored. From this, we conclude that in TRβ-/- mice,

the presence of SRC-1 inhibits TRα-mediated suppression of GST, and when SRC-1 is also

removed, the TRα, possibly acting with another coregulatory molecule, mediates TH-induced

GST suppression.

Of the TH responsive genes identified to be SRC-1-dependent, we were unable to

confirm the microarray data with RT PCR for these 2 genes. In WT mice, SHSF increased by

40% without any response in SRC-1-/- mice (Table VII). However there were also blunted

responses to TH in the other genotypes investigated, indicating that SHSF will not be a good

E-00226-2002.R2

22

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 TRβ-/- mice to TH,

showing increases in ERF expression (Table VII), versus decreases seen by microarray (Table

VI). 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 TRβ-dependent markers

(Osteopontin and GST) in the presence of TH. These genes have not been previously identified

in 2 other studies of TH responsive gene expression (8, 9). We have seen other genes reported

by microarray that were not reproduced by other methods (unpublished data) and note that the

previous studies only confirmed 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 liver

expression of 5’DI by RT PCR, an enzyme whose expression has been known to be up-regulated

by thyroid hormone via the TRβ (1). Interestingly, we saw sharp increases in 5’DI expression in

TRα0/0 mice, far greater than in any other genotype (Fig. 3). Macchia et al recently reported that

mice deficient in all known TRα isoforms have hypersensitivity to thyroid hormone (22). It was

speculated that a potential mechanism for hypersensitivity in these animals would be elimination

of the inhibitory TRα2. However, as 5’DI data would indicate, the hypersensitivity conferred

upon TRα-deficient mice (evidenced by vast TH-induced increases in 5’DI expression) is

ablated in the absence of both TRα and SRC-1, in which TRα0/0SRC-1-/- mice increase 5’DI in

response to TH to similar levels as WT and SRC-1-/- animals. This result indicates that the

hypersensitivity seen in TRα0/0 mice may be due to more than absence of the inhibitory TRα2.

In fact, it is likely that the hypersensitivity is due to an increased availability of SRC-1 to interact

E-00226-2002.R2

23

with the TRβ, an event that may be controlled in TR-competent mice by squelching of the

coactivator by the TRα.

A model of TH action in liver is shown in figure 4. 5’deiodinase represents a liver gene

up-regulated by TH. TRβ1 appears to be the key isoform to mediate TH action on 5’DI in the

liver, as in its absence, there is a major reduction in 5’DI induction with TH. In TRα0/0 mice,

there is a hyperresponse in 5’DI to TH. We propose that this TH hypersensitivity is due to relief

of inhibition by TRα2, as originally hypothesized by Macchia et al (22). However, we further

this hypothesis to include that SRC-1 is necessary for this action in liver, and that SRC-1 inhibits

TRα2 activity, as TRα0/0SRC-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 observing

the effect on gene expression. Additionally, we propose that SRC-1 facilitates TRβ-induced

5’DI expression, but is not necessary (Figure 5B). It is possible that in the absence of SRC-1,

alternative coactivators, such as TIF-2 or SRC-3 could compensate.

From these studies, we conclude that (1) in the absence of TRα or TRβ, SRC-1 is

important for normal growth; (2) SRC-1 partially mediates the TH effect on heart rate by TRα

and TRβ; (3) we have identified two new TH responsive markers in the liver mediated by TRβ;

osteopontin (up-regulated) and glutathione-S-transferase (down-regulated); and (4) we have

shown that SRC-1 may mediate the hypersensitivity to TH seen in TRα0/0 mice as demonstrated

by 5’deoiodinase expression in liver.

E-00226-2002.R2

24

Acknowledgements

The authors are indebted to Prof. Samuel Refetoff for advise and guidance during this project

and Dr. T. Karrision for help with statistical analyses. This work was supported in part by grants

from the National Institutes of Health DK 58281 (to R.E.W.) the Ministry of Research ACI 283 (to

J.S.), DK 58242 (to J.X.), and NICHD 078587 (to B.O’M.) and by the Seymour J. Abrams Thyroid

Research Center. The authors gratefully acknowledge the technical assistance of Kevin Cua with

preliminary energy expenditure and heart rate experiments.

E-00226-2002.R2

25

Figure Legends

Figure 1. Growth of mice of genotypes from two weeks until adulthood. Length of mice of

each genotype was determined from age 2 weeks to age 9 weeks (above). Body weight was

determined for the same time period of time. Comparisons are shown for WT, TRβ-/-, SRC-1-/-,

and TRβ-/-SRC-1-/- mice on the left (A, C) and WT, TRα0/0, SRC-1-/-, TRα0/0SRC-1-/- on the right

(B, D). Numbers of animals in each group (N) is shown in the legend. Values in the curves

represent the mean of each group ± SE. Where no SE is not present, values were sufficiently low

as to not appear using the graphing program. A table of significance is shown below the graphs.

Comparison made between WT mice and other genotypes at 5 weeks and 9 weeks by Tukey-

Kramer method at 5% confidence.

Figure 2. Relationship of serum T4 levels and energy expenditure (EE) (kcal/day/gm body

weight), (top); and nonfasting serum Leptin (ng/ml/gm body weight), (bottom). Values represent

means. Note that EE and Leptin and T4 levels were the highest in the TRβ-/-SRC-1-/- mice.

Figure 3. mRNA quantitation of 5’Deiodinase in liver. 5’Deiodinase expression was

determined by TaqMan quantitative real time PCR. Data is shown as percent increase (on a

logarithmic scale) from PTU-treated wildtype when treated with four days ip injection of T3

(0.2µg/25g/day). The percent increase is determined from the mean liver expression of these

genes from at least five mice in each treatment group and each genotype. SE determined from %

change of individual samples from PTU-treated WT mice. Five mice were used for each

hypothyroid and T3-treated group.

Figure 4. Model for thyroid hormone action in the liver. Arrows represent activation and

blockers represent inhibition.Thickness or lines or number of arrows represents potential strength

of interaction.

E-00226-2002.R2

26

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Tab

le I

. G

enes

up

and

dow

n re

gula

ted

by T

H in

TR

β-/- a

nd S

RC

-1-/

-m

ice*

.G

enot

ype

#Gen

es R

espo

ndin

g (%

)#U

p R

egul

ated

(%

)#D

own

Reg

ulat

ed (

%)

WT

92

(7.8

)77

(83

.7)

15 (

16.3

)T

Rβ-/

-99

(8.

4)34

(34

.2)

65 (

65.6

)SR

C-1

-/-

105

(8.9

)97

(92

.3)

8 (

7.6)

*Thr

esho

ld r

atio

s of

1.6

7 w

ere

used

for

eac

h co

mpa

riso

n

TA

BLE

II.

Pro

be/P

rimer

Set

s fo

r T

aqM

an Q

uant

itativ

e R

eal T

ime

PC

R u

sed

for

mR

NA

qua

ntita

tion.

GE

NE

prim

ers

sequ

ence

sor

ient

atio

n

5'-TGCCTCCTCCCTCCCGGTGA

-3'

sens

e*O

steo

pont

in5'

-GATTTGCTTTTGCCTGTTTGG

-3'

sens

e5'

-TGAGCTGCCAGAATCAGTCACT

-3'

an

tisen

se

5'-CGTGCTTCACTACTTCAATGCCCGG

-3'

sens

e*G

ST

5'-GCAATGGCCGGGAAGC

-3'

sens

e5'

-ACCTGATGCACTCCATTCTGC

-3'

antis

ense

5'-TCTTGGCGCGGCGCATGTC

-3'

sens

e*S

HS

F5'

-GAGGTCCGAGCAGCTTGG

-3'

sens

e5'

-AAGTCGACCGGCTGCTTCTT

-3'

antis

ense

5'-CCAGCACGCCACAGCCCAACT

-3'

sens

e*E

RF

5'-CCACGACGGGTGAGCTCT

-3'

se

nse

5'-ATAGCTCTCAGGAATCTCGGTGC

-3'

antis

ense

5'-CTGCCTGAGAGGCTCTACGTGATACAGGA

-3'

sens

e*5’

DI

5'-AGCCAGCTCTACGCGGC

-3'

se

nse

5'-CCCTTGTAGCAGATCCTGCC

-3'

antis

ense

*FA

M/T

AM

RA

fluo

resc

ent T

aqM

an P

robe

(M

egaB

ases

, Eva

nsto

n, IL

).

Tab

le I

II.

Thy

roid

fun

ctio

n te

sts.

BA

SEL

INE

PT

U/L

oI1

PT

U/L

oI +

L-T

32

GE

NO

TY

PE

TT

4*T

T3*

TSH

*T

SH*

TSH

*

µg/d

lng

/dl

mU

/lm

U/l

mU

/l

Wild

type

3.7

± 0

.1 (

53)

73

± 5

(21

) 3

0 ±

5 (

65)

93

11 ±

811

(16

)

4

6 ±

21

(16)

TR

β-/-

7.6

± 0

.3 (

53)**

139

± 9

(23

)**25

8 ±

44

(57)

**

1491

6 ±

1,3

06 (

21) *

*

2941

± 3

01 (

17) *

*

TR

α0/0

3.2

± 0

.1 (

33) *

*96

± 6

(12

) 2

3 ±

4 (

34)

4754

± 5

29 (

17) *

*

3

2 ±

10

(18)

** SRC

-1-/

- 4

.7 ±

0.2

(19

) **

130

± 4

(12

) **

74

± 1

9 (1

9) *

*92

76 ±

840

(24

)

966

± 3

27 (

18) *

*

TR

β-/-SR

C-1

-/-

12.6

± 2

.6 (

11) *

*40

4 ±

39

(7) *

*73

3 ±

152

(19

) **

2346

8 ±

1,4

40 (

18) *

* 1

2645

± 1

,290

(12

) **

TR

α0/0 SR

C-1

-/-

6.5

± 0

.3 (

18) *

* 1

59 ±

8 (

14) *

* 5

2 ±

12

(18)

**

52

58 ±

591

(19

) **

10

25 ±

449

(12

) **

Mea

n ±

sta

ndar

d er

ror.

Num

bers

in p

aren

thes

es =

num

ber

of m

ice

1 Low

Iod

ine/

PTU

die

t for

14

days

2 LoI

/PT

U d

iet 1

4 da

ys w

ith I

P L

-T3 0

.2µg

/25g

bod

y w

t/day

*, O

ne-w

ay A

NO

VA

for

eff

ect o

f ge

noty

pe o

n th

yroi

d te

st e

xam

ined

, p<

0.00

01

**si

gnif

ican

ce a

t 5%

by

Tuk

ey/K

ram

er c

ompa

red

to w

ildty

pe

Table IV. Baseline Energy Expenditure and Leptin

Genotype Energy Expenditure(EE/day/g body wt)* Leptin (ng/mL/gm body wt)*

Wildtype 0.477 ± 0.010 (42) 94 ± 6 (6)

TRα0/0 0.458 ± 0.017 (33) 94 ± 13 (6)

TRα0/0SRC-1-/- 0.495 ± 0.015 (33) 165 ± 23 (11)

SRC-1-/- 0.487 ± 0.017 (32) 204 ± 43** (15)

TRβ-/-SRC-1-/- 0.667 ± 0.025 (9)** 236 ± 47** (9)

TRβ-/- 0.492 ± 0.013 (27) 158 ± 21 (12)

(N) Number of mice.*, One-way ANOVA for effect of genotype p<0.0001; ** significantdifference at 5% compared to wildtype by Tukey/Kramer.

Table V. Heart Rates of Mice at Baseline, Hypothyroid, and Hypothyroid with T3

Beats/Minute ± SEGenotype

No Tx* 14 days PTU* PTU+0.2µgT3* % Change w/T3*

Wildtype 524 ± 10 (20) 405 ± 12 (24) 537 ± 19 (7) 32.4 ± 4.7 (7)TRα0/0 371 ± 28 (11) 319 ± 10 (25) 407 ± 33 (7) 27.7 ± 10.4 (7)TRα0/0SRC-1-/- 351 ± 16 (14) 323 ± 19 (19) 380 ± 12 (6) 17.6 ± 3.8 (6)

SRC-1-/- 388 ± 13 (19) 292 ± 11 (27) 418 ± 15 (13) 43.2 ± 5.0 (13)TRβ-/-SRC-1-/- 483 ± 20 (20) 353 ± 16 (9) 520 ± 14 (5) 47.1 ± 4.1 (5)TRβ-/- 472 ± 16 (20) 340 ± 19 (20) 466 ± 23 (17) 37.0 ± 6.6 (17)

Tukey/Kramer 5% Sig Mice on Normal Diet

WT TRα0/0 TRβ-/- SRC-1-/- TRα0/0

SRC-1-/-

TRβ-/-

SRC-1-/-

WT SIG NS SIG SIG NS

TRα0/0 SIG SIG NS NS SIG

TRβ-/- NS NS SIG SIG NS

SRC-1-/- SIG NS NS NS SIG

TRα0/0

SRC-1 -/- SIG NS NS NS SIG

TRβ -/-SRC-1-/- NS NS NS NS SIG

Mice on LoI/PTU Diet + 0.2µgT3/25g/day* One-way ANOVA examining effect of genotype, p<0.0001; SIG, significant; NS, notsignificant

Tab

le V

I. G

enes

iden

tifi

ed t

o be

up

or d

own

regu

late

d in

res

pons

e to

TH

and

dep

ende

nt o

n ei

ther

TR

or

SRC

-1.

G

enba

nk #

(Nam

e)E

ffec

t of

TH

WT

TR

-/-

SRC

-1-/

-

TR

Dep

ende

nt

J048

06 (

oste

opon

tin)

↑2.8

no e

ffec

t↑2

.0

J039

58 (

glut

athi

one

s-tr

ansf

eras

e)↓3

.5no

eff

ect

↓4.3

SRC

-1 D

epen

dent

U

4160

6 (s

plit

hand

/foo

t gen

e)↑2

.4↑2

.7no

eff

ect

U

5853

3 (E

ts-r

elat

ed tr

ans.

Fac

tor)

↓1.8

↓2.4

no e

ffec

t

Tab

le V

II.

Fold

indu

ctio

n w

ith L

-T3 t

reat

men

t of

vari

ous

gene

s m

easu

red

by T

aqM

an P

CR

(N

=5

anim

als

in e

ach

grou

p).

Gen

otyp

eO

steo

pont

inG

luta

thio

ne-S

-Tra

nsfe

rase

Split

Han

d Sp

lit F

oot

Ets

-Rel

ated

Fac

tor

Wild

type

1.86

± 0

.39*

0.21

± 0

.02*

1.45

± 0

.08*

1.59

± 0

.22*

TR

α0/0

0.89

± 0

.11∆

0.31

± 0

.04*

1.14

± 0

.04∆

0.96

± 0

.16

TR

β-/-

1.02

± 0

.13∆

1.34

± 0

.16∆

1.17

± 0

.09

1.9

± 0

.38*

SRC

-1-/

-1.

36 ±

0.1

6*0.

19 ±

0.0

4*1.

19 ±

0.0

91.

3 ±

0.2

1

TR

α0/0 /S

RC

-1-/

-0.

84 ±

0.0

6∆0.

25 ±

0.0

5*1.

03 ±

0.0

5∆0.

76 ±

0.1

1

TR

β-/- /S

RC

-1-/

-1.

00±

0.1

7∆0.

23 ±

0.0

4*0.

91 ±

0.0

2∆1.

01 ±

0.1

1

AN

OV

Ap

Val

ue0.

0062

<0.

0001

<0.

0001

0.00

35

*: 5

% s

igni

fica

nce

com

pare

d to

PT

U tr

eatm

ent,

sam

e ge

noty

pe∆:

5%

sig

nifi

canc

e co

mpa

red

to L

-T3 t

reat

men

t, w

ildty

pe

Figure 1

Age (Weeks)

5

10

15

20

25

30

2 3 4 5 6 7 8 95

10

15

20

25

30

2 3 4 5 6 7 8 9

TRβ-/- (15)

TRβ-/-SRC-1-/- (6)SRC-1-/- (8)

Wildtype (10)

TRα0/0SRC-1-/- (10)

5

6

7

8

9

10

11

2 3 4 5 6 7 8 95

6

7

8

9

10

11

2 3 4 5 6 7 8 9

A

Statistics: Tukey-KramerCompare Wildtype to: Length Weight Length Weight

SIG SIG SIG NSSIG NS SIG NSSIG NS NS NSSIG SIG SIG SIGSIG SIG SIG SIG

SIG, significance at 5% confidence; NS=Not significant; One-way ANOVA, p<0.0001 for effect of genotype on length and weight at each time point

TRα0/0

TRβ-/-

5 weeks 9 weeks

SRC-1-/-

TRα0/0 SRC-1-/-

TRβ-/- SRC-1-/-

C

B

D

TRα0/0 (5)

[T4]

0.4

0.5

0.6

0.7

4 6 8 10 12 14

WTTRβ-/-

TRαo/oSRC-1-/-

TRβ-/-/SRC-1-/-

TRαo/oSRC-1-/-

WT

TRβ-/-

TRαo/o

SRC-1-/-TRβ-/-/SRC-1-/-

TRαo/oSRC-1-/-

0

100

200

300

Fig. 2

WT TR 0/0 TR -/-SRC-1-/-TR 0/0

SRC-1-/-TR -/-

SRC-1-/-

Figure 3

1

10

100

1000

10000

LIV

ER

SR

C-1

TR

TR

T3

500%

WIL

DT

YP

E

LIV

ER

SR

C-1

TR

TR

T3

1500

%T

R0/

0

LIV

ER

SR

C-1

TR

TR

T38%

TR

-/-

LIV

ER

SR

C-1

TR

TR

T3

500%

SR

C-1

-/-

TIF

-2?

SR

C-3

?

Fig

. 4