help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0239
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kamiya, Y.
Right arrow Articles by Cheng, S.-Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kamiya, Y.
Right arrow Articles by Cheng, S.-Y.
Endocrinology Vol. 144, No. 9 4144-4153
Copyright © 2003 by The Endocrine Society

Modulation by Steroid Receptor Coactivator-1 of Target-Tissue Responsiveness in Resistance to Thyroid Hormone

Yuji Kamiya, Xiao-Yong Zhang, Hao Ying, Yusuhito Kato, Mark C. Willingham, Jianming Xu, Bert W. O’Malley and Sheue-Yann Cheng

Laboratory of Molecular Biology (Y.K., X.-Y.Z., H.Y., Y.K., S.-Y.C.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264; Department of Pathology (M.C.W.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157; and Department of Cell Biology (J.X., B.W.O.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. S.-Y. Cheng, Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Room 5128, Bethesda, Maryland 20892-4264. E-mail: sycheng{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutations in the thyroid hormone receptor-ß gene (TRß) cause resistance to thyroid hormone. How the action of mutant thyroid hormone nuclear receptors (TRs) is regulated in vivo is not clear. We examined the effect of a TR coactivator, steroid receptor coactivator-1 (SRC-1), on target-tissue responsiveness by using a mouse model of resistance to thyroid hormone, TRßPV knockin mice, in the SRC-1 null background. Lack of SRC-1 intensified the dysfunction of the pituitary-thyroid axis and impaired growth in TRßPV/+ mice but not in TRßPV/PV mice. In TRßPV/PV mice, however, lack of SRC-1 intensified the pathological progression of thyroid follicular cells to papillary hyperplasia, reminiscent of papillary neoplasia. In contrast, lack of SRC-1 did not affect responsiveness in the liver in regulating serum cholesterol in either TRßPV/+ or TRßPV/PV mice. Lack of SRC-1 led to changes in the abnormal expression patterns of several T3 target genes in the pituitary and liver. Thus, the present studies show that a coactivator such as SRC-1 could modulate the in vivo action of TRß mutants in a tissue-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE THYROID HORMONE T3 promotes growth, induces differentiation, and regulates metabolic functions. These biological activities are mediated by the interaction of T3 with thyroid hormone nuclear receptors (TRs) that are members of the steroid/retinoic acid receptor superfamily. Two TR genes, TR{alpha} and TRß, located on human chromosomes 17 and 3, respectively, have been identified. Four T3-binding TR isoforms, ß1, ß2, ß3, and {alpha}1, are generated from these two genes (1, 2). Each TR isoform can be functionally divided into three domains: the amino-terminal A/B domain, the DNA-binding domain C, and the carboxy-terminal ligand-binding domain E. Domain C recognizes specific hormone response DNA sequences in the promoter region of T3 target genes, whereas domain E, besides binding T3, participates in transcriptional activation and repression. The gene-regulating activity of TRs is complex. It depends not only on T3 and the types of thyroid hormone response elements but also on other cellular proteins including coactivators and corepressors (3).

Resistance to thyroid hormone (RTH) is a syndrome characterized by the resistance of tissues to the action of thyroid hormone. This disease is manifested by elevated levels of circulating thyroid hormone and dysfunction of the negative feedback loop in the pituitary-thyroid axis, resulting in insuppressible TSH. Clinical features include goiter, attention-deficit hyperactivity disorder, decreased IQ, dyslexia, short stature, decreased weight, tachycardia, cardiac disease, and hypercholesterolemia (4). RTH can appear sporadically, but most commonly it is a familial syndrome with autosomal dominant inheritance. RTH is caused by mutations in TRß (4). The mutations are clustered in three hot spots in the hormone-binding domain (4, 5). TRß mutants derived from RTH patients have reduced or no T3-binding activity and transcriptional capacities. They act in a dominant negative fashion to cause the clinical phenotype (4, 6).

To study the molecular basis of RTH, we created an RTH mouse model by targeting the PV mutation to the TRß gene locus via homologous recombination (TRßPV mice) (7). The PV mutation was derived from a patient with severe RTH characterized by attention-deficit hyperactivity disorder, short stature, low weight, goiter, and tachycardia. PV has a unique mutation in exon 10, a C-insertion at the coding nucleotide position 1642 (codon 448) that produces a frame shift in the carboxy-terminal 14 amino acids of TRß1, resulting in total loss of T3 binding and transcriptional activities. The pituitary-thyroid axis is mildly impaired in the heterozygous TRßPV/+ mice. In homozygous TRßPV/PV mice, the severe dysfunction of the pituitary-thyroid axis results in an extraordinarily high TSH level despite highly elevated levels of circulating thyroid hormone. TRßPV/PV mice also have impaired weight gain and delayed bone development (7). This phenotype is entirely consistent with that of human RTH, and, therefore, TRßPV/PV mice represent a valid model of human RTH.

One of the intriguing observations in RTH patients is a marked variability of resistance in different tissues of the same individual or among kindreds with the same TRß gene mutations (4, 6). How the action of mutant TRß is modulated, leading to varying degrees of resistance in tissues, is not clear. The availability of the TRßPV mouse provides an unusual opportunity to address this question. Using these mice, we recently found that one of the factors that contribute to variability in tissue sensitivity of RTH is the differential distribution of endogenous TR isoforms in tissues (8). However, differential TR isoform distribution alone is not sufficient to fully account for the abnormal expression patterns of T3 target genes in different tissues (8).

Recent studies indicate that the transcriptional activity of wild-type TRs is regulated by coregulatory proteins (3). In the absence of T3, TR represses basal transcriptional activity by binding to corepressors, such as the nuclear corepressor or the silencing mediator of retinoic and thyroid hormone receptor, that are associated with histone deacetylases via an intermediary factor, Sin3 (9, 10). Binding of T3 results in dissociation of this corepressor complex from TR, enabling TR to recruit coactivators [e.g. steroid receptor coactivator-1 (SRC-1) and/or other p160 family members] and other cofactors associated with histone acetyltransferase activity, leading to transcriptional activation (3). Indeed, the activation role of SRC-1 has also been shown in vivo in that mice with wild-type receptors, but deficient in SRC-1, exhibit the phenotype of reduced T3 (11) and steroid hormone insensitivity (12). The reduced T3 sensitivity in tissues because of the lack of SRC-1 is TR-subtype dependent (13). However, whether the lack of SRC-1 in vivo affects tissue resistance because of TRß mutants is currently unknown.

In the present study, we asked whether the lack of SRC-1 alters the extent of responsiveness in the pituitary-thyroid axis and impairs growth and bone development, as manifested in TRßPV mice (7). We also evaluated the effect of lack of SRC-1 on the abnormal expression patterns of several T3 target genes in the pituitary and liver. We found that the lack of SRC-1 modulates the degree of responsiveness to thyroid hormone in a tissue-dependent manner. Moreover, the present findings show an expanded role for SRC-1 in modulating not only the functions of wild-type TRs but also actions of TRß mutants in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse strains
All animal experiments were performed according to protocols approved by the Animal Care and Use Committee at NIH. The TRßPV/PV mice and SRC-1-/- mice were generated as described previously (7, 12). The TRßPV/+ and SRC-1-/+ mice were crossed, and the resultant offspring were backcrossed to each other five or six times to generate offspring with genotypes of TRß+/+SRC-1+/+, TRß+/+SRC-1-/-, TRßPV/+SRC-1+/+, TRßPV/+SRC-1-/-, TRßPV/PVSRC-1+/+, and TRßPV/PVSRC-1-/- mice. They were genotyped by PCR using primers specific for TRß and SRC-1 sequences as described (7, 12). The littermates were used in the comparison of the phenotypes among mice with different genotypes. Mice were fed normal chow and tap water and housed under 12-h light/12-h dark cycles at 22 C. For Northern analysis of CYP7A mRNA in liver, mice were fed fat-free test diet (ICN Biomedicals, Costa Mesa, CA) for 5 d before being killed.

Hormone assays
Sera were collected from adult mice (2–5 months old). Serum levels of total T4 and T3 were determined by using a GammaCoat T4 or T3 assay RIA kit (DiaSolin, Stillwater, MN) according to the manufacturer’s instructions. TSH levels in serum were measured as previously described (7, 14) but using the RIA kit purchased from National Hormone and Peptide Program, Harbor-UCLA Medical Center (Torrance, CA). Sera from overnight-fasted mice were used for determination of total cholesterol levels in accordance with the directions provided by the test kit manufacturer (cholesterol, total, and cholesterol calibrator kits; Sigma, St. Louis, MO).

Northern blot analysis
Total RNA from three mice (3 months old) was prepared as described for Northern blot analysis (7). After electrophoresis, RNA was transferred onto membranes (Hybond-N+; Amersham Pharmacia, Boston, MA), which were hybridized with appropriate probes. The cDNA clones for TSHß, GH, and cholesterol 7{alpha}-hydroxylase (CYP7A) were labeled with [{alpha}-32P]dCTP by using a Prime It II (Stratagene Cloning Systems, La Jolla, CA). For quantification, the intensities of the mRNA bands were normalized against the intensities of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (n = 3). The blots were stripped and rehybridized with a 32P-labeled GAPDH cDNA. Quantification was performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) or NIH Image 1.61.

Histological examination
Thyroid glands were fixed in 10% neutral buffered formalin and subsequently embedded in paraffin. Five-micrometer-thick sections were prepared and stained with hematoxylin and eosin for microscopic examination.

The areas of individual follicles were determined by using Photoshop software (version 7.0, Adobe, San Jose, CA) with the histogram function that allows the measurement of areas in pixels at a fixed magnification and digital resolution. Random selection of follicles (n = 100) was facilitated by the use of the magic wand tool in Photoshop, set at a tolerance that selected the entire follicle, including epithelial cells.

The morphological determination of the normal, hyperplastic, and papillary hyperplastic follicular cells was performed, first by identification of the normal follicular cells. The remaining cells constituted a group termed hyperplastic cells. Further subgrouping identified the hyperplastic cells that had at least one single frond. This subgroup was termed papillary hyperplastic cells. A microscope equipped with high-resolution objectives was used to identify the hyperplastic follicular cells with or without fronds and fibrovascular stalks (papillary hyperplastic cells). Multiple samples with multiple observations were used to make the determinations. For consistency, blinded samples were examined by a single pathologist using the same criteria.

Data analysis
All data are expressed as mean ± SE. P values were calculated using one-way ANOVA. Follow-up tests were performed using the Fisher’s protected least square difference. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of SRC-1 intensifies the dysfunction of the pituitary-thyroid axis in TRßPV mice
Thyroid hormone production is tightly regulated by a feedback mechanism in that pituitary TSH is suppressed by elevated levels of thyroid hormone. To understand the role of SRC-1 in the regulation of the pituitary-thyroid axis, we first compared the circulating levels of thyroid hormone in wild-type and TRßPV mice with or without SRC-1 (Fig. 1Go). Lack of SRC-1 resulted in mean total T4 (TT4) that was 1.7-fold higher (TRß+/+SRC-1+/+ = 4.9 ± 0.3 µg/dl, n = 10; TRß+/+SRC-1-/- = 8.3 ± 0.5 µg/dl, n = 18). Consistent with findings in RTH patients who have a mutated TRß gene (7, 11), mean TT4 was 2.2-fold higher in TRßPV/+SRC-1+/+ mice (11.0 ± 0.8 µg/dl, n = 13) than TRß+/+ mice. Importantly, lack of SRC-1 in TRßPV/+SRC-1-/- mice led to an additional 2.1-fold increase in TT4 (23.40 ± 0.8 µg/dl, n = 20). There were, however, no significant differences in the highly elevated mean TT4 levels between the TRßPV/PVSRC-1+/+ and TRßPV/PVSRC-1-/- mice (83.7 ± 7.2 µg/dl, n = 10 and 96.5 ± 5.4 µg/dl, n = 20, respectively; P = 0.115).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 1. Comparison of serum thyroid hormones in TRßPV mice with or without SRC-1. Serum levels of TT4 (A) and serum levels of TT3 (B) in adult mice (2–3 months old). Each circle represents the value for an individual mouse and the horizontal bars represent the mean values. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

 
Lack of SRC-1 in wild-type and TRßPV mice resulted in a similar effect on the total T3 (TT3) levels (Fig. 1BGo). Mice deficient in SRC-1 (TRß+/+SRC-1-/- mice) had a mean TT3 concentration (2.1 ± 0.1 ng/ml, n = 15) that was 1.5-fold that of wild-type mice (1.33 ± 0.05 ng/ml, n = 10). Consistent with observations in RTH patients who have a mutated TRß gene (7, 11), the mean TT3 concentration was elevated 1.7-fold in TRßPV/+SRC-1+/+ mice (2.3 ± 0.1 ng/ml, n = 12), compared with TRß+/+ mice. The lack of SRC-1 in TRßPV/+SRC-1-/- mice further increased the mean TT3 concentration 2.0-fold (4.5 ± 0.3 ng/ml, n = 15). The highly elevated mean TT3 concentration in TRßPV/PVSRC-1+/+ mice (20.0 ± 3.4 ng/ml, n = 10) was not significantly different from that in TRßPV/PVSRC-1-/- mice (28.3 ± 4.2 ng/ml, n = 15; P = 0.106). These results indicate that lack of SRC-1 increased the circulating levels of thyroid hormones in TRßPV/+ mice but not significantly in TRßPV/PV mice. The lack of effect of the absence of SRC-1 on TT4 and TT3 could be because the response has reached its maximum in the presence of two mutated TRß genes.

The extent of thyroid gland enlargement correlated with the magnitude of the elevation of serum thyroid hormone concentration in TRßPV mice. As shown in Fig. 2Go, the mean thyroid weight of TRß+/+SRC-1-/- mice (4.2 ± 0.2 mg, n = 10) was 1.5-fold that of TRß+/+SRC-1+/+ mice (2.8 ± 0.3 mg, n = 11). The mean thyroid weight of TRßPV/+SRC-1+/+ mice (5.4 ± 0.3 mg, n = 10) was 1.9-fold that of TRß+/+SRC-1+/+ mice. Importantly, mean thyroid weight of TRßPV/+SRC-1-/- mice (10.5 ± 0.8 mg, n = 11) was also 1.9-fold that of TRßPV/+SRC-1+/+ mice. The lack of SRC-1 did not significantly affect the degree of enlargement of thyroid glands in TRßPV/PVSRC-1+/+ vs. TRßPV/PVSRC-1-/- mice (41.6 ± 5.2 mg, n = 10, vs. 57.8 ± 10.5 mg, n = 10, P = 0.08).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 2. Comparison of the weights of thyroid glands of TRßPV mice with or without SRC-1. The weights of thyroid glands from 3-month-old mice were determined. Each circle represents the value for an individual mouse and the horizontal bars represent the mean values. **, P < 0.01; ***, P < 0.001; NS, not significant.

 
Consistent with the weight increase in the thyroids, histological evaluation indicated that lack of SRC-1 significantly increased the size of the individual follicles. The average area of the follicles of TRß+/+SRC-1-/- mice (Fig. 3BGo) was 2358 ± 178 pixels/follicle (n = 100; mean ± SD), which was 1.7-fold that of TRß+/+SRC-1+/+ mice (1420 ± 27 pixels/follicle; Fig. 3AGo). The average area of follicles of TRßPV/+SRC-1-/- mice (Fig. 3DGo) was 4817 ± 716 pixels/follicle, which was 1.6-fold that of TRßPV/+SRC-1+/+ mice (3015 ± 319 pixels/follicle; Fig. 3CGo). More dramatic changes were detected in the thyroids of TRßPV/PV mice. The epithelium of the thyroids in TRßPV/PVSRC-1+/+ mice exhibited extensive hyperplasia (Fig. 3EGo). Lack of SRC-1 in TRßPV/PVSRC-1-/- mice intensified the pathological progression of the thyroid gland to produce florid adenomatous hyperplasia with a papillary pattern and papillary containing fibrovascular cores, reminiscent of papillary neoplasia (Fig. 3FGo).



View larger version (68K):
[in this window]
[in a new window]
  		FIG. 3. Representative histological features of thyroid glands of TRßPV mice with or without SRC-1. Hematoxylin- and eosin-stained sections of routinely formaldehyde-fixed, paraffin-embedded thyroid glands from 3-month-old TRß+/+SRC-1+/+ mice (A); TRß+/+SRC-1-/- mice (B); TRßPV/+SRC-1+/+ mice (C); TRßPV/+SRC-1-/- mice (D); TRßPV/PVSRC-1+/+ mice (E); and TRßPV/PVSRC-1-/- mice (F). G, Distribution of the histopathological patterns of follicles in thyroids of TRßPV/PVSRC-1+/+ and TRßPV/PVSRC-1-/- mice. One hundred follicles were scored on randomly selected areas of thyroid section in each mouse, according to Materials and Methods. A total of eight mice were examined in each genotype.

 
Because the follicles of the thyroid glands in TRßPV/PVSRC-1+/+ (Fig. 3EGo) and TRßPV/PVSRC-1-/- (Fig. 3FGo) mice were heterogeneous, we developed a way to quantify the degree of pathological progression. We characterized each thyroid according to the percentage of the follicles that was normal, hyperplastic but without papillary structure, or hyperplastic with papillary pattern. As shown in Fig. 3GGo, the majority (~75%) of the follicles in TRßPV/PVSRC-1+/+ mice were hyperplastic (bar 3, Fig. 3GGo), whereas the majority (~60%) in TRßPV/PVSRC-1-/- mice were hyperplastic with papillary patterns (bar 6, Fig. 3GGo), an indication that the lack of SRC-1 intensified the pathological progression in the epithelium of the thyroids in TRßPV/PVSRC-1-/- mice.

To understand the effect of SRC-1 on the function of the pituitary, we determined the serum TSH levels. Figure 4Go shows that despite elevated thyroid hormone, there was no significant difference between the serum TSH level in TRßPV/+SRC-1+/+ mice (56.0 ± 7.6 ng/ml, n = 17) and that in TRß+/+SRC-1+/+ mice (43.4 ± 5.2 ng/ml, n = 12), indicating the resistance of the pituitary to the action of thyroid hormone. Significantly, the TSH level in TRßPV/+SRC-1-/- mice (104.9 ± 10.1 ng/ml, n = 29) was 1.9-fold that in TRßPV/+SRC-1+/+ mice. Compared with the wild-type mice, mice without the mutated TRß gene but lacking SRC-1 (TRß+/+SRC-1-/-, n = 13) had a 1.9-fold higher serum TSH level. Lack of SRC-1 (TRßPV/PVSRC-1-/- mice; n = 28) did not significantly increase the extraordinarily high TSH in TRßPV/PVSRC-1+/+ mice (n = 18; P = 0.198). The lack of effect of SRC-1 on TSH could be because the response has reached its maximum in the presence of two mutated TRß genes. However, the extent of the increased weight of thyroid glands correlated with the elevated TSH levels.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 4. Comparison of serum TSH levels in TRßPV mice with or without SRC-1. Serum TSH levels of adult mice (2–5 months old). Each circle represents the value for an individual mouse and the horizontal bars represent the mean values. *, P < 0.05; ***, P < 0.001; NS, not significant.

 
Lack of SRC-1-impaired growth in TRßPV mice
Consistent with previous results (7), female TRßPV/PVSRC-1+/+ mice (Fig. 5CGo vs. 5AGo) had impaired weight gain. TRßPV/PVSRC-1+/+ mice weighed significantly less than their wild-type littermates at all ages examined. In contrast, no significant difference in weight gain was detected between TRß+/+SRC-1+/+ mice and TRßPV/+SRC-1+/+ mice (Fig. 5BGo vs. 5AGo). Interestingly, lack of SRC-1 led to impaired weight gain in TRßPV/+SRC-1-/- mice (Fig. 5BGo). A significantly lower weight gain (9–12% lower) was observed up to 10 wk of age for TRßPV/+SRC-1-/- mice, compared with their TRßPV/+SRC-1+/+ littermates (P < 0.05). Importantly, the lack of SRC-1 did not significantly affect the weight gain in mice without the mutation in the TRß gene (Fig. 5AGo). In fact, after wk 8, TRß+/+SRC-1-/- mice tended to gain significantly more weight (~6% increase at 12 wk of age) than did TRß+/+SRC-1+/+ mice. The lack of SRC-1, however, did not significantly affect the growth/weight gain of TRßPV/PVSRC-1-/- mice (Fig. 5CGo). Similar results were found for male mice (data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5. Comparison of weekly weight gains of TRßPV mice with or without SRC-1. The weights of female TRßPV mice with ({bullet}) or without SRC-1 (o) were measured in mice 4–12 wk old. Data are shown as mean ± SE (n = 12–15).

 
In a separate study in which the pups were weighed beginning at the age of 1 wk, no significant differences in weight gains between TRßPV/+SRC-1+/+ and TRßPV/+SRC-1-/- mice were detected until the age of 4 wk. The significant differences in weight gain between TRß+/+SRC-1+/+ and TRßPV/PVSRC-1+/+ were found at the age of 3 wk and thereafter. No significant differences in body lengths were found (our unpublished data).

Lack of SRC-1 does not alter the resistance of the liver in the regulation of serum cholesterol
To understand whether the lack of SRC-1 alters the responsiveness of the liver to thyroid hormone, we measured the serum cholesterol that is an in vivo marker of thyroid hormone action. Thyroid hormone is known to lower cholesterol levels. The blunted response in serum cholesterol after administration of thyroid hormone is commonly used as a diagnostic test for assessing RTH in peripheral tissues (6). Figure 6Go shows that, compared with the wild-type mice (bar 1; n = 14), TRßPV/+SRC-1+/+ mice exhibited the phenotype of resistance in the liver by failing to properly respond to the elevated level of thyroid hormone, as indicated by no repression of the cholesterol levels (bar 3; n = 19). Lack of SRC-1 did not significantly change the total cholesterol levels in mice without PV mutation (bar 2; n = 19; P = 0.694) or in mice with one PV allele (bar 4; n = 19; P = 0.151). More dramatic resistance of the liver to the action of thyroid hormone was detected in TRßPV/PVSRC-1+/+ mice in that their serum cholesterol levels were significantly higher than those of TRß+/+SRC-1+/+ mice (Fig. 6Go, bar 5 vs. bar 1; 1.35-fold; n = 19). Lack of SRC-1, however, did not further increase the cholesterol levels in TRßPV/PVSRC-1-/- mice (an increase of 1.44-fold; bar 6 vs. bar 1, n = 19; P = 0.343).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 6. Comparison of serum total cholesterol levels in TRßPV mice with or without SRC-1. Serum total cholesterol levels were determined from adult mice (2–3 months old), as described in Materials and Methods. The data are expressed as mean ± SE (n = 14–20). ***, P < 0.001; NS, not significant.

 
Lack of SRC-1 lessens impairment of bone development in TRßPV/PV mice
Figure 7Go shows that lack of SRC-1 did not significantly affect the development of the tibia in mice without the mutation of the TRß gene (18.42 ± 0.19 mm, n = 11 and 17.93 ± 0.33 mm, n = 6 for TRß+/+SRC-1+/+ mice and TRß+/+SRC-1-/- mice, respectively; P = 0.19) or in mice with one PV allele (17.94 ± 0.14 mm, n = 10 and 18.21 ± 0.15 mm, n = 10 for TRßPV/+SRC-1+/+ mice and TRßPV/+SRC-1-/- mice, respectively; P = 0.537). Unexpectedly, we found that the lack of SRC-1 significantly lessened impairment of tibial growth in TRßPV/PV mice (16.96 ± 0.14 mm, n = 9 and 17.58 ± 0.08 mm, n = 7 for TRßPV/PVSRC-1+/+ mice and TRßPV/PVSRC-1-/- mice, respectively; P < 0.05).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 7. Comparison of the lengths of tibia in TRßPV mice with or without SRC-1. The lengths of tibia were determined in adult mice (3–4 months old). Each circle represents the value for an individual mouse and the horizontal bars represent the mean values. *, P < 0.05; ***, P < 0.001; NS, not significant.

 
Lack of SRC-1 intensifies the abnormal expression patterns of TSHß and GH genes in the pituitary
To gain insight into how SRC-1 modulates the target-tissue responsiveness in TRßPV mice, we examined the effect of the lack of SRC-1 on the expression patterns of T3-target genes in tissues. We first examined the expression of the TSHß gene in TRßPV mice. As shown in Fig. 8Go, the lack of SRC-1 did not significantly change the expression of the TSHß gene in TRß+/+ mice (Fig. 8A-aGo, lanes 1–3 vs. 4–5, and Fig. 8BGo, bar 2 vs. bar 1, for TRß+/+SRC-1+/+ and TRß+/+SRC-1-/-, respectively; P = 0.241). Consistent with our previous report (7), the expression of the TSHß gene was not repressed despite elevated thyroid hormones in TRßPV/+SRC-1+/+ mice. The lack of SRC-1 accentuated the resistance to thyroid hormone in the pituitary by a 1.4-fold increase in the expression of the TSHß mRNA (Fig. 8BGo, bar 4 vs. 3; P < 0.05). Although the expression of the TSHß mRNA was greatly augmented in TRßPV/PVSRC-1+/+ (Fig. 8BGo, bar 5 vs. bar 1) and TRßPV/PVSRC-1-/- mice (Fig. 8BGo, bar 6 vs. bar 2), the lack of SRC-1 did not significantly affect the expression of the TSHß gene in TRßPV/PV mice (Fig. 8BGo, bar 6 vs. bar 5; P = 0.362). These results indicate that the effect of the lack of SRC-1 on the expression of the TSHß mRNA is consistent with that of the serum levels of TSH (see Fig. 4Go).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 8. Comparison of the expression of the TSHß and GH genes in the pituitary of TRßPV mice with or without SRC-1. Northern blot analysis of TSHß mRNA (A-a) and GH mRNA (A-b) were determined using 3 µg/lane of total RNA from three adult male mice. The loading of RNA was normalized by stripping the blots and rehybridizing with [32P]-labeled GAPDH (A-c). After quantification, the relative mRNA expression is shown for TSHß (B) and for GH (C). Data are expressed as mean ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

 
The expression of the GH gene is positively regulated by T3. Figures 8A-bGo and 8CGo show that instead of being activated by elevated thyroid hormone in TRßPV/+SRC-1+/+ mice, the expression of the GH mRNA had no increase, compared with that in TRß+/+SRC-1+/+ mice (Fig. 8CGo, bar 3 vs. bar 1). No changes in the expression of the GH mRNA because of the lack of SRC-1 were detected between TRßPV/+SRC-1+/+ and TRßPV/+SRC-1-/- mice (Fig. 8CGo, bar 4 vs. bar 3), indicating that the lack of SRC-1 did not further enhance the dominant negative effect of PV on the expression of the GH mRNA. A strong repression of GH mRNA (80% reduction) was observed in TRßPV/PVSRC-1+/+ mice (Fig. 8CGo, bar 5 vs. bar 1). An additional small reduction (~10%) was observed in TRßPV/PV mice deficient in SRC-1 (Fig. 8CGo, bar 6 vs. bar 5). As shown in Fig. 8A-cGo, the expression of GAPDH was used to normalize the expression of the TSHß mRNA in Fig. 8A-aGo and the GH mRNA in Fig. 8A-bGo.

Lack of SRC-1 does not alter the PV-mediated abnormal expression pattern of the CYP7A gene in TRßPV mice
T3 is important not only for the uptake of cholesterol by the liver but also for the hepatic degradation of cholesterol into bile acids. The rate-limiting enzyme in the latter process, CYP7A, is activated by T3 at the transcriptional level and is therefore a direct T3 positively regulated gene (15, 16). We, therefore, evaluated the effect of SRC-1 on the expression of CYP7A mRNA in TRßPV mice. Figure 9Go shows that no activation was detected in TRßPV/+SRC-1+/+ mice (Fig. 9BGo, bar 3 vs. bar 1) in the face of elevated serum thyroid hormone. The lack of SRC-1 did not change the expression of the CYP7A gene in the liver of either TRß+/+SRC-1-/- mice (Fig. 9BGo, bar 2 vs. bar 1) or TRßPV/+SRC-1-/- mice (Fig. 9BGo, bar 4 vs. bar 3).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 9. Comparison of the expression of the CYPA7 gene in the liver of TRßPV mice with or without SRC-1. Northern blot analysis of the CYPA7 mRNA (A-a) was determined using 20 µg/lane of total RNA from the livers from three adult male mice. The loading of RNA was normalized by stripping the blots and rehybridizing with [32P]-labeled GAPDH (A-b). After quantification, the relative mRNA expression is shown (B). Data are expressed as mean ± SE. *, P < 0.05; NS, not significant.

 
In TRßPV/PVSRC-1+/+ mice, instead of being activated by the highly elevated thyroid hormone (see Fig. 1Go, A and B), the expression of CYP7A mRNA was repressed 80% (TRßPV/PVSRC-1-/- mice; Fig. 9BGo, bar 5 vs. bar 1). This extent of repression was not altered in mice deficient in SRC-1 (Fig. 9BGo, bar 6 vs. bar 5; P = 0.979). These results indicate that, consistent with findings from serum cholesterol levels (Fig. 6Go), the expression the CYP7A gene mediated by PV is not affected by the lack of SRC-1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that the lack of SRC-1 led to an array of changes in the phenotypic expression of TRßPV mice. These changes can be roughly classified into three groups. The first group of changes relates to patterns found in the pituitary-thyroid axis and in growth/weight gain. Namely, lack of SRC-1 enhanced resistance in TRßPV/+ mice but had no significant effect on TRßPV/PV mice. Thus, serum TT4, TT3, and TSH levels and the weight of thyroid glands in TRßPV/+ mice were markedly increased in mice deficient in SRC-1. In TRßPV/+SRC-1+/+ mice in which no manifestation of abnormality in weight gain was detected, compared with TRß+/+SRC-1+/+ mice, the lack of SRC-1 led to the manifestation of impairment in growth. However, no enhancement in these two phenotypes was found in TRßPV/PV mice by the lack of SRC-1. The second group of changes is exemplified in the liver in that no alterations in serum total cholesterol levels were detected by the lack of SRC-1 in either TRßPV/+ or TRßPV/PV mice. The third group is illustrated by the impairment of bone development in that the lack of SRC-1 had no significant effect on the extent of the impaired bone development in TRßPV/+ mice but lessened the extent of impairment in TRßPV/PV mice. These results suggest that the effect of the lack of SRC-1 on resistance is target tissue dependent. However, we cannot exclude the possibility that the complex phenotype exhibited by TRßPV/PVSRC-1-/- mice could be from the contributions of other coregulatory proteins because of secondary effects from the lack of SRC-1 (e.g. possible compensatory effect).

Recently, on the basis of studies using mice deficient in TR{alpha} and SRC-1 (TR{alpha}0/0SRC-1-/-) as well as in TRß and SRC-1 (TRß-/-SRC-1-/-), Sadow et al. (13, 17) reported that SRC-1 also modulates the in vivo actions of wild-type TRs in several T3 target tissues. The lack of SRC-1 increases the extent of resistance in the pituitary-thyroid axis of TR-/- mice in that an additional 1.6-, 2.9-, and 2.8-fold elevation in TT4, TT3, and TSH serum concentrations, respectively, were detected (15). A similar but slightly lower additional increase in TT4, TT3, and TSH concentrations caused by the lack of SRC-1 (1.7-, 1.7-, and 2.2-fold, respectively) in TR{alpha}0/0SRC-1-/- mice was also observed. No impairment in weight gain is apparent in mice deficient in either TRß or SRC-1, but the lack of SRC-1 led to the manifestation of impaired weight gain in TRß-/-SRC-1-/- mice (13). However, the lack of SRC-1 does not further increase the extent of impaired weight gain already exhibited in TR{alpha}0/0 mice (13). In the heart, the lack of SRC-1 significantly lowers the basal heart rates in mice with the presence of both TR isoforms (SRC-1-/- mice), but no further significant changes were discernible in TRß-/-SRC-1-/- mice. A relatively small (1.4-fold), but significant, additional lowering of the heart rates in TR{alpha}0/0 mice was observed along with the lack of SRC-1 (13). These studies demonstrate that, similar to what was found in TRßPV mice, SRC-1 affects the in vivo activity of TRs in a tissue-dependent manner. The present findings highlight an expanded role of SRC-1 in that it not only modulates the in vivo functions of wild-type TRs (11, 12, 13, 17) but also plays a critical role in the action of mutant TRß in vivo.

To understand how the lack of SRC-1 alters the target tissue responsiveness, we ascertained the effect of the lack of SRC-1 on the expression of several T3-target genes. Previously it was shown that the expression of the TSHß gene in the pituitary was not repressed in TRßPV/+ mice, notwithstanding increased thyroid hormone (7). The present study shows that the lack of SRC-1 further increased the expression of TSHß mRNA (1.4-fold) in TRßPV/+ mice. That SRC-1 plays a functional role in the increased expression of the TSHß gene is supported by the studies in TRß-/-SRC-1-/- mice (17). In mice deficient in either TRß or SRC-1, a 3.3- or 1.3-fold increase in the expression of the TSHß mRNA was detected. The lack of SRC-1 (TRß-/-SRC-1-/- mice) led to an 8.9-fold increase in the expression of the TSHß mRNA (17).

In mice with two mutated TRß genes, however, the lack of SRC-1 did not significantly increase further the already elevated TSHß mRNA (Fig. 8AGo). One possibility we propose is that the difference in the effect of the lack of SRC-1 on the expression of TSHß mRNA is due to the number of the mutated TRß alleles. In TRßPV/+ mice, the lack of SRC-1 could compromise the transcriptional activity of the remaining wild-type TRß allele in the pituitary of TRßPV/+ mice, thereby further increasing the abnormal expression of the TSHß gene. This notion is supported by the DNA-binding experiments reported earlier in that, in addition to the detection of TRß/PV heterodimers, TRß/RXR heterodimers were also detected in TRßPV/+ mice (8). In the presence of T3 and SRC-1, TRß/RXR heterodimers presumably mediate the normal function of repressing the expression of the TSHß gene. Therefore, in TRßPV/+SRC-1+/+ mice, only one mutated TRß allele acts to interfere the transcriptional activity of the wild-type TRs (one copy of the TRß gene and two copies of the TR{alpha} genes), leading to mild abnormal expression of the TSHß gene.

However, the lack of SRC-1 weakens the normal functions of wild-type TRß and TR{alpha}1, thereby resulting in increased abnormal regulation of the TSHß gene in TRßPV/+SRC-1-/- mice. In TRßPV/PVSRC-1-/- mice, both the TRß genes are mutated. TRß is known to be the predominant TR isoform in the pituitary (18). Because the expression of PV is driven by the same promoter as the TRß gene, the more abundantly expressed PV (compared with TR{alpha}1) exerted a strong dominant negative effect on the abnormal up-regulation of the TSHß gene that was not significantly affected by the lack of SRC-1 in the pituitary of TRßPV/PV mice. This finding suggests that the weakening effect of SRC-1 on the wild-type TR{alpha}1 plays a minor role in the regulation of the TSHß gene. This notion is supported by the findings in the expression of the TSHß mRNA in TRß-/- and TR{alpha}0/0 mice. The lack of TRß has a more severe effect on the dysregulation of the TSHß gene than does the lack of TR{alpha}1 (19, 20, 21). These results indicate the critical role of the interplay of a differential expression of TR isoforms and SRC-1 in the manifestation of resistance in target tissues.

With a limited number of T3 target genes that could be evaluated, there was a good correlation in the effect of the lack of SRC-1 between the mRNA expression levels of TSHß and the serum levels of TSH. In the liver, there was also a good correlation between the expression of the CYP7A gene and the serum cholesterol levels in that the lack of SRC-1 had no effect on either the PV-mediated abnormal expression patterns of the CYP7A gene or the elevated cholesterol levels. However, no correlation in the effect of the lack of SRC-1 on the expression of the GH gene with the growth/weight gain was observed. Namely, in the TRßPV/+SRC-1-/- mice, the lack of SRC-1 did not change the expression of the GH gene, whereas the lack of SRC-1 did lead to the manifestation of growth retardation. In TRßPV/PV mice, the PV-mediated repression of the GH gene was further intensified, but no further impairment in growth/weight gain was observed in the absence of SRC-1. These results suggest that PV-mediated growth impairment requires the participation of other genes that are yet to be uncovered.

The present studies demonstrate that the target-tissue resistance is subject to multifactor combinatorial regulation. The interplay of tissue-dependent TR isoform distribution, coactivators such as SRC-1, and the environment of local promoters of T3-target genes leads to variable tissue resistance to thyroid hormone, as demonstrated in TRßPV mice deficient in SRC-1. Recently a large number of coregulatory proteins have been isolated (3, 22). Some are general and some are tissue and/or receptor specific. This would mean that there are numerous combinatorial possibilities in the modulation of tissue responsiveness to thyroid hormone. Thus, it should not be a surprise that kindred with the same TRß mutation manifest different degrees of resistance in the same tissues or exhibit different spectra of resistance in different tissues within the same patient. With the lessons learned from the TRßPV mouse model, it should be possible to design better strategies to treat RTH patients.


   Acknowledgments
 
We thank Dr. Hideyo Suzuki for sharing the unpublished data on the weigh gains of TRßPV mice at early ages.


   Footnotes
 
Y.K. was supported in part by the Japan Society for the Promotion of Science Research Fellowship for Japanese Biomedical and Behavioral Research at the National Institutes of Health. B.W.O’M. and J.X. were supported by NICHD Grant 07857.

Abbreviations: CYP7A, Cholesterol 7{alpha} hydroxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RTH, resistance to thyroid hormone; SRC-1, steroid hormone receptor coactivator-1; TR, thyroid hormone nuclear receptor; TRß, thyroid hormone receptor ß subtype; TRßPV, TRß mutant PV; TT3, total T3; TT4, total T4.

Received February 21, 2003.

Accepted for publication May 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cheng SY 2000 Multiple mechanisms for regulation of the transcriptional activity of thyroid hormone receptors. Rev Endocr Metab Disord 1:9–18[CrossRef][Medline]
  2. Williams G 2000 Cloning and characterization of two novel thyroid hormone receptor ß isoforms. Mol Cell Biol 20:8329–8342[Abstract/Free Full Text]
  3. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465–474[CrossRef][Medline]
  4. Weiss RE, Refetoff S 2000 Resistance to thyroid hormone. Rev Endocr Metab Disord 1:97–108[CrossRef][Medline]
  5. Collingwood TN, Wanger R, Matthews CH, Clifton-Bligh RJ, Gurnell M, Rajanayagam O, Agostini M, Fletterick RJ, Beck-Peccoz P, Reinhardt W, Binder G, Ranke M, Hermus A, Hesch RD, Lazarus J, Newrick P, Parfitt V, Raggatt P, Zegher FD, Chatterjee VKK 1998 A role of helix 3 of the TRß ligand-binding domain in coactivator recruitment identified by characterization of a third cluster of mutation in resistance to thyroid hormone. EMBO J 17:4760–4770[CrossRef][Medline]
  6. Refetoff S, Weiss RE, Usala SJ 1993 The syndromes of resistance to thyroid hormone. Endocrinol Rev 14:348–399[Abstract/Free Full Text]
  7. Kaneshige M, Kaneshige K, Zhu XG, Dace A, Garrett L, Carter TA, Kazlauskaite R, Pankrat DG, Wynshaw-Boris A, Refetoff S, Weintraub B, Willingham MC, Barlow C, Cheng SY 2000 Mice with a targeted mutation in the thyroid hormone ß receptor gene exhibit impaired growth and resistance to thyroid hormone. Proc Natl Acad Sci USA 97:13209–13214[Abstract/Free Full Text]
  8. Zhang XY, Kaneshige M, Kamiya Y, Kumiko K, McPhie P, Cheng SY 2002 Differential expression of thyroid hormone receptor isoform dictates the dominant negative activity of mutant ß receptor. Mol Endocrinol 16:2077–2092[Abstract/Free Full Text]
  9. Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD, Torchia J, Yang WM, Brard G, Ngo SD, Davie JR, Seto E, Eisenman RN, Rose DW, Glass CK, Rosenfeld MG 1997 A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387:43–48[CrossRef][Medline]
  10. Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE, Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by a complex containing SMRT, mSin3A and histone deacetylase. Cell 89:373–380[CrossRef][Medline]
  11. Weiss RE, Xu J, Ning G, Pohlenz J, O’Malley BW, Refetoff S 1999 Mice deficient in the steroid receptor co-activator 1 (SRC-1) are resistant to thyroid hormone. EMBO J 18:1900–1904[CrossRef][Medline]
  12. Xu J, Qiu Y, DeMayo FJ, Tsai SY, Tsai MJ, O’Malley BW 1998 Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 279:1922–1925[Abstract/Free Full Text]
  13. Sadow PM, Chassande O, Gauthier K, Samarut J, Xu J, O’Malley BW, Weiss R 2003 Specificity of thyroid hormone receptor subtype and steroid receptor coactivator-1 on thyroid hormone action. Am J Physiol Endocrinol Metab 284:E36–E46
  14. Kaneshige M, Suzuki H, Kaneshige K, Cheng J, Wimbrow H, Barlow C, Willingham MC, Cheng S-Y 2001 A targeted dominant negative mutation of the thyroid hormone {alpha}1 receptor causes increased mortality, infertility and dwarfism in mice. Proc Natl Acad Sci USA 98:15095–15100[Abstract/Free Full Text]
  15. Crestani M, Karam WG, Chiang JY 1994 Effects of bile acids and steroid/thyroid hormones on the expression of cholesterol 7{alpha}-hydroxylase mRNA and the CYP7 gene in HepG2 cells. Biochem Biophys Res Commun 198:546–553[CrossRef][Medline]
  16. Pandak WM, Heuman DM, Redford K, Stravitz RT, Chiang JY, Hylemon PB, Vlahcevic ZR 1997 Hormonal regulation of cholesterol 7{alpha}-hydroxylase specific activity, mRNA levels, and transcriptional activity in vivo in the rat. J Lipid Res 38:2483–2491[Abstract]
  17. Sadow PM, Koo E, Chassande O, Gauthier K, Samarut J, Xu J, O’Malley BW, Seo H, Murata Y, Weiss RE 2003 Thyroid hormone receptor-specific interactions with steroid receptor coactivator-1 in the pituitary. Mol Endocrinol 17:882–894[Abstract/Free Full Text]
  18. Hsu JH, Brent GA 1998 Thyroid hormone receptor knockouts. Trends Endocrinol Metab 9:103–112
  19. Forrest D, Hanebuth E, Smeyne RJ, Everds N, Stewart CL, Wehner JM, Curran T 1996 Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor ß: evidence for tissue-specific modulation of receptor function. EMBO J 15:3006–3015[Medline]
  20. Forrest D, Vennstrom B 2000 Functions of thyroid hormone receptors in mice. Thyroid 10:41–52[Medline]
  21. Gauthier K, Chassande O, Plateroti M, Roux JP, Legrand C, Pain B, Rousset B, Weiss R, Trouillas J, Samarut J 1999 Different functions for the thyroid hormone receptors TR{alpha} and TPß in the control of thyroid hormone production and post-natal development. EMBO J 18:623–631[CrossRef][Medline]
  22. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465–474



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. L. Privalsky, S. Lee, J. B. Hahm, B. M. Young, R. N. G. Fong, and I. H. Chan
The p160 Coactivator PAS-B Motif Stabilizes Nuclear Receptor Binding and Contributes to Isoform-specific Regulation by Thyroid Hormone Receptors
J. Biol. Chem., July 17, 2009; 284(29): 19554 - 19563.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. J. O'Shea, C. J. Guigon, G. R. Williams, and S.-y. Cheng
Regulation of Fibroblast Growth Factor Receptor-1 (Fgfr1) by Thyroid Hormone: Identification of a Thyroid Hormone Response Element in the Murine Fgfr1 Promoter
Endocrinology, December 1, 2007; 148(12): 5966 - 5976.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. N. Winnay, J. Xu, B. W. O'Malley, and G. D. Hammer
Steroid Receptor Coactivator-1-Deficient Mice Exhibit Altered Hypothalamic-Pituitary-Adrenal Axis Function
Endocrinology, March 1, 2006; 147(3): 1322 - 1332.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
H. Ying, F. Furuya, M. C. Willingham, J. Xu, B. W. O'Malley, and S.-y. Cheng
Dual Functions of the Steroid Hormone Receptor Coactivator 3 in Modulating Resistance to Thyroid Hormone
Mol. Cell. Biol., September 1, 2005; 25(17): 7687 - 7695.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
E. Setiawan, D. Owen, L. McCabe, A. Kostaki, M. H. Andrews, and S. G. Matthews
Glucocorticoids Do Not Alter Developmental Expression of Hippocampal or Pituitary Steroid Receptor Coactivator-1 and -2 in the Late Gestation Fetal Guinea Pig
Endocrinology, August 1, 2004; 145(8): 3796 - 3803.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kamiya, Y.
Right arrow Articles by Cheng, S.-Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kamiya, Y.
Right arrow Articles by Cheng, S.-Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals