TRbeta mRNA Research Articles

Thyroid disruption induced by pentachlorophenol

To assess thyroid disruption induced by sodium pentachlorophenol (PCP) using Organization for Economic Co-operation and Development (OECD) recommended TG 407 method. A total of 30 specific pathogen free (SPF) SD adult male and female rats were randomly divided into 3 groups, and treated with water, 0.33 and 30 mg x kg(-1)x d(-1) of PCP-Na by oral gavage for consecutive 28 days, respectively. After final treatment, histological changes of thyroid were observed by hematoxylin-eosin stain, and the levels of thyroid hormones (total thyroxine (TT(4)), free thyroxine (FT(4)), total triiodothyronine (TT(3)), and free triiodothyronine (FT(3))) were determined by radioimmunoassay. The expression levels of thyroid receptors (TRalpha and TRbeta) mRNA and deiodinases (DioI, DioII and DioIII) mRNA in liver were analyzed by RT-PCR. In high dose group, liver weight coefficient of male and female rats were (4.82 +/- 0.42)% and (4.99 +/- 0.17)%, increased by 36.2% (t = 7.338, P < 0.01) and 41.8% (t = 8.955, P < 0.01), compared to control group ((3.54 +/- 0.14)%, (3.52 +/- 0.19)%), respectively, while the significant changes of kidney or thyroid weight were not observed. In high dose group, the levels of TT(4) and FT(4) in serum of male rats were (64.95 +/- 7.16) nmol/L and (8.16 +/- 2.29) pmol/L, and decreased by 26.6% (t = -3.999, P < 0.01) and 42.3% (t = -4.112, P < 0.01) compared to control group ((88.48 +/- 6.99) nmol/L, (14.13 +/- 1.68) pmol/L). In the same group, FT(4) in serum of female rats was (4.94 +/- 0.89) pmol/L, decreased by 55.5% (t = -3.380, P = 0.012) compared to control group ((11.10 +/- 3.40) pmol/L) and TT(3) and FT(3) in serum of female rats were (1.92 +/- 0.24) nmol/L and (3.05 +/- 0.79) pmol/L, increased by 74.5% (t = 5.263, P < 0.01) and 55.6% (t = 3.495, P < 0.01) compared to control group ((1.10 +/- 0.23) nmol/L, (1.96 +/- 0.32) pmol/L), respectively. PCP-Na didn't affect the expression levels of TRalpha, TRbeta, DioIII mRNA in high dose group, while DioII expression of male rats (0.209 +/- 0.017) down-regulated by 79.2% (t = -5.426, P < 0.01) compared to control group (1.006 +/- 0.137), and DioI expression of female rats (1.844 +/- 0.189) up-regulated by 66.6% (t = 4.359, P < 0.01) compared to control group (1.005 +/- 0.083), indicating DioI and DioII poss different sensitivity to adverse effects induced by PCP-Na between male and female rats. The histopathological results showed that PCP-Na could give rise to hyperplasia of the follicular epithelium cells, and the depletion of colloid. There were no significant changes in serum THs levels and expression of TRalpha, TRbeta, DioI-IIImRNA in low dose group. However, sporadic lymphocytic infiltration, follicles amplification in part and slightly increased in thickness of follicular cells were observed in this group. PCP is a kind of thyroid disrupting chemical.

Seasonal variations in testicular connexin levels and their regulation in the brook trout, Salvelinus fontinalis

Spermatogenesis requires coordinated intercellular communication mediated by gap junctions. Gap junctions are composed of connexons that are themselves composed of connexins (Cxs). The present objective was to determine the regulation of testicular Cxs in a seasonal breeder, the brook trout. To assess seasonal variations in testicular Cxs, trout were sampled monthly throughout spermatogenesis (June–November). Circulating levels of testosterone (T) and 11-ketotestosterone (11-KT) as well as mRNA levels for testicular androgen receptors ( ar-alpha, ar-beta), thyroid hormone receptors ( tr-alpha, tr-beta) and gonadotropin I receptor ( rgthI) were measured. Plasma T levels peaked in October, one month prior to spawning, while 11-KT levels peaked at spawning. ar-alpha and ar-beta mRNA levels increased during spermatogenesis and peaked in November while tr-alpha, tr-beta mRNA levels stayed constant throughout spermatogenesis and increased dramatically in November . rgthI mRNA levels decreased progressively during spermatogenesis. Cx43 and Cx30 levels were constant during spermatogenesis and decreased in November. Cx31 levels were also constant during spermatogenesis but decreased dramatically in October and November. Cx43.4 levels peaked in July then decreased in September and levels were undetectable thereafter. Using in vitro cultures of testicular fragments we demonstrated that cx43 mRNA levels were regulated in a dose–response manner by 3,5,3′-triiodo- l-thyronine (0–370 nM) and cAMP (0–100 ng/ml) but levels were not regulated by 11-KT. These results indicate that testicular Cxs vary as a function of spermatogenesis and that the expression of cx43 in the trout testis is regulated by both cAMP and TH.

The Thyroid Hormone Receptor (TR) β-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR β mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta1 over TRalpha1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TRalpha1 than TRbeta1 mRNA in rat (approximately 20-90%) and mouse (approximately 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TRalpha1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TRbeta1 mRNA expression increases (approximately 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TRbeta1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TRbeta1 mRNA expression to a similar extent in both cell lineages (approximately 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TRbeta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Thyroid Hormone Receptor-β Is Associated with Coronary Angiogenesis during Pathological Cardiac Hypertrophy

Insufficient angiogenesis is one of the causes leading to tissue ischemia and dysfunction. In heart failure, there is increasing evidence showing decreased capillary density in the left ventricle (LV) myocardium, although the detailed mechanisms contributing to it are not clear. The goal of this study was to investigate the role of thyroid hormone receptors (TRs) in the coronary microvascular rarefaction under pathological cardiac hypertrophy. The LV from hypertrophied/failing hearts induced by ascending aortic constriction (AAC) exhibited severe microvascular rarefaction, and this phenomenon was restored by chronic T(3) administration. Coronary endothelial cells (ECs) isolated from AAC hearts expressed lower TRbeta mRNA than control ECs, and chronic T(3) administration restored TRbeta mRNA expression level in AAC hearts to the control level. Among different TR subtype-specific knockout mice, TRbeta knockout and TRalpha/TRbeta double-knockout mice both exhibited significantly less capillary density in LV compared with wild-type mice. In vitro, coronary ECs isolated from TRbeta knockout mice lacked the ability to form capillary networks. In addition, we identified that kinase insert domain protein receptor/fetal liver kinase-1 (vascular endothelial growth factor-2 receptor) was one of the angiogenic mediators controlled by T(3) administration in the AAC heart. These data suggest that TRbeta in the coronary ECs regulates capillary density during cardiac development, and down-regulation of TRbeta results in coronary microvascular rarefaction during pathological hypertrophy.

Genistein prevents thyroid hormone‐dependent tail regression of Rana catesbeiana tadpoles by targetting protein kinase C and thyroid hormone receptor α

Thyroid hormone (TH)-regulated gene expression is mainly mediated by TH binding to nuclear thyroid hormone receptors (TRs). Despite extensive studies in mammalian cell lines that show that phosphorylation signaling pathways are important in TH action, little is known about their roles on TH signaling in vivo during development. Anuran metamorphosis is a postembryonic process that is absolutely dependent upon TH and tadpole tail resorption can be precociously induced by exogenous administration of 3,5,3'-triiodothyronine (T(3)). We demonstrate that genistein (a major isoflavone in soy products and tyrosine kinase inhibitor) and the PKC inhibitor (H7) prevent T(3)-induced regression of the Rana catesbeiana tadpole tail. T(3)-induced protein kinase C tyrosine phosphorylation and kinase activity are inhibited by genistein while T(3)-induced up-regulation of TRbeta mRNA, but not TRalpha mRNA, is significantly attenuated, most likely through inhibition of T(3)-dependent phosphorylation of the TRalpha protein. This phosphorylation may be modulated through PKC. These data demonstrate that T(3) signaling in the context of normal cells in vivo includes phosphorylation as an important factor in establishing T(3)-dependent tail regression during development.

Expression of Thyroid Hormone Receptor Isoforms Down-regulated by Thyroid Hormone in Human Medulloblastoma Cells

The role of thyroid hormone (T3) in the regulation of growth and development of the central nervous system including the cerebellum has been well established. However, the effects of thyroid hormone on malignant tumors derived from the cerebellum remain poorly understood. Our analysis mainly focused on expression levels of TR isoforms and the effects of thyroid hormone in human medulloblastoma HTB-185 cells. Northern blot analysis revealed TRalpha2 mRNA but not TRalpha1, beta1 or beta2 mRNA in the cell. The TRalpha1 and TRbeta1 mRNAs were detected only by RT-PCR method and TRbeta2 was not expressed. Incubation of T3 for 24 h decreased TRalpha1, TRalpha2 and TRbeta1 mRNA. Addition of actinomycin D caused an acute increase in the basal TR mRNA levels and the rate of decrease of all kinds of TR isoform mRNA was accelerated in the T3-treated groups compared to controls, indicating that the stability of TR mRNA was affected by T3. Incubation with cycloheximide also blocked a decrease in TR mRNA levels in the T3-treated HTB-185 cells suggesting that down-regulation of TR mRNA required the synthesis of new protein. Our data provide novel evidence for the expression of TRs down-regulated by T3 in HTB-185 cells, suggesting that TR expression is post-transcriptionally regulated by T3 at the level of RNA stability.

The Use of Real‐Time PCR to Study the Expression of Thyroid Hormone Receptor β2 in the Developing Chicken

Thyroid hormones and their receptors (TRs) have critical functions in development and metabolism. In chicken, three TRs are known: TRalpha, TRbeta0, and TRbeta2. The latter was isolated from chicken eye, but its presence in other tissues has not yet been extensively investigated. We therefore developed a real-time PCR assay using a Taqman probe and primers based on the unique amino-terminal region of TRbeta2. We detected a strong TRbeta2 mRNA signal in the pituitary, confirmed with in situ hybridization, and in several other tissues. TRbeta2 mRNA was more abundant in the pituitary of newly hatched chicks than in 15-day-old embryos.

Expression of thyroid hormone receptor alpha in 3T3-L1 adipocytes; triiodothyronine increases the expression of lipogenic enzyme and triglyceride accumulation.

Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRalpha and TRbeta, which act as mediators of thyroid hormone in tissues. However, the relative expression of each TR isoform in target tissues is still elusive. Herein, we have developed an RT-PCR and restriction enzyme digestion method to determine the expression of TRalpha1 and TRbeta1. We analyzed the expression of TR isoforms in 3T3-L1 preadipocytes induced to differentiate by an adipogenic cocktail in the presence or absence of 100 nM triiodothyronine (T(3)). The TRalpha1 isoform was predominantly expressed in 3T3-L1 adipocytes, and its expression was increased at the stage of development concomitant with the emergence of lipid droplets. Little, if any, TRbeta1 mRNA was detected in adipocytes. Administration of T(3) to the differentiating 3T3-L1 cells enhanced the accumulation of triglyceride. The expression profile of TRalpha1 in T(3)-treated adipocytes was similar to that in non-treated cells. The transcripts of adipogenic factors, CCAAT/enhancer binding protein beta (C/EBPbeta) and peroxisome proliferator activated receptor gamma (PPARgamma), were not altered by T(3). Lipid binding protein, aP2, that is downstream of these transcription factors was also unaffected by T(3). In contrast, the lipogenic enzyme, glyceraldehyde-3-phosphate dehydrogenase mRNA was significantly increased in the presence of T(3). Therefore, T(3) appears to be a hormone capable of modulating the expression of lipogenic enzyme and augments the accumulation of lipid droplets. We conclude that the TRalpha isoform might play an important role in the generation and maintenance of the mature adipocyte phenotype, regulating the expression of lipogenic enzymes.

Simultaneous changes in central and peripheral components of the hypothalamus-pituitary-thyroid axis in lipopolysaccharide-induced acute illness in mice.

During illness, major changes in thyroid hormone metabolism and regulation occur; these are collectively known as non-thyroidal illness and are characterized by decreased serum triiodothyronine (T(3)) and thyroxine (T(4)) without an increase in serum TSH. Whether alterations in the central part of the hypothalamus-pituitary-thyroid (HPT) axis precede changes in peripheral thyroid hormone metabolism instead of vice versa, or occur simultaneously, is presently unknown. We therefore studied the time-course of changes in thyroid hormone metabolism in the HPT axis of mice during acute illness induced by bacterial endotoxin (lipopolysaccharide; LPS).LPS rapidly induced interleukin-1beta mRNA expression in the hypothalamus, pituitary, thyroid and liver. This was followed by almost simultaneous changes in the pituitary (decreased expression of thyroid receptor (TR)-beta2, TSHbeta and 5'-deiodinase (D1) mRNAs), the thyroid (decreased TSH receptor mRNA) and the liver (decreased TRbeta1 and D1 mRNA). In the hypothalamus, type 2 deiodinase mRNA expression was strongly increased whereas preproTRH mRNA expression did not change after LPS. Serum T(3) and T(4) fell only after 24 h. Our results suggested almost simultaneous involvement of the whole HPT axis in the downregulation of thyroid hormone metabolism during acute illness.

Developmental expression and hormonal regulation of glucocorticoid and thyroid hormone receptors during metamorphosis in Xenopus laevis.

Corticosteroids, the primary circulating vertebrate stress hormones, are known to potentiate the actions of thyroid hormone in amphibian metamorphosis. Environmental modulation of the production of stress hormones may be one way that tadpoles respond to variation in their larval habitat, and thus control the timing of metamorphosis. Thyroid hormone and corticosteroids act through structurally similar nuclear receptors, and interactions at the transcriptional level could lead to regulation of common pathways controlling metamorphosis. To better understand the roles of corticosteroids in amphibian metamorphosis we analyzed the developmental and hormone-dependent expression of glucocorticoid receptor (GR) mRNA in the brain (diencephalon), intestine and tail of Xenopus laevis tadpoles. We compared the expression patterns of GR with expression of thyroid hormone receptor beta (TRbeta). In an effort to determine the relationship between nuclear hormone receptor expression and levels of ligand, we also analyzed changes in whole-body content of 3,5,3'-triiodothyronine (T(3)), thyroxine, and corticosterone (CORT). GR transcripts of 8, 4 and 2 kb were detected in all tadpole tissues, but only the 4 and 2 kb transcripts could be detected in embryos. The level of GR mRNA was low during premetamorphosis in the brain but increased significantly during prometamorphosis, remained at a constant level throughout metamorphosis, and increased to its highest level in the juvenile frog. GR mRNA level in the intestine remained relatively constant, but increased in the tail throughout metamorphosis, reaching a maximum at metamorphic climax. The level of GR mRNA was increased by treatment with CORT in the intestine but not in the brain or tail. TRbeta mRNA level increased in the brain, intestine and tail during metamorphosis and was induced by treatment with T(3). Analysis of possible crossregulatory relationships between GRs and TRs showed that GR mRNA was upregulated by exogenous T(3) (50 nM) in the tail but downregulated in the brain of premetamorphic tadpoles. Exogenous CORT (100 nM) upregulated TRbeta mRNA in the intestine. Our findings provide evidence for tissue-specific positive, negative and crossregulation of nuclear hormone receptors during metamorphosis of X. laevis. The synergy of CORT with T(3) on tadpole tail resorption may depend on the accelerated accumulation of GR transcripts in this tissue during metamorphosis, which may be driven by rising plasma thyroid hormone titers.

Expression of thyroid hormone receptor isoform alpha1 in pancreatic islets.

Thyroid hormone receptors (TR) mediate the action of thyroid hormones. Genetic studies revealed that the individual TR isoforms possess different functions. In the present paper we studied the expression of the isoforms TRalpha1 and TRbeta1 in the murine pancreatic islet. TRalpha1 and TRbeta1 mRNA transcripts and proteins were detected in islets using reverse transcription-polymerase chain reaction and Western blotting analyses, respectively. In immunohistochemical studies individual cells in the periphery of islets were labelled using an anti-TRalpha1 antibody. No labelled cells were detected in the exocrine pancreas. A similar staining pattern was obtained with an anti-glucagon antibody, but not with an anti-insulin antibody, which suggests that TRalpha1 is mainly expressed in alpha-cells. In order to address a potential function of TRalpha1 in this cell type, the regulation of glucagon gene expression by triiodothyronine was studied in a glucagon-producing cell line by Northern blot analysis and transient transfection assays using glucagon promoter luciferase fusion gene constructs. In these assays, triiodothyronine did not regulate the glucagon mRNA level or the glucagon promoter activity. The predominant localization of TRalpha1 in pancreatic alpha-cells suggests that this receptor isoform mediates a specific, yet unknown, function of thyroid hormones in this cell type.

Thyroid hormone responsiveness in N-Tera-2 cells.

N-TERA-2 cl/D1 (NT2) cells, a human embryonal cell line with characteristics of central nervous system precursor cells, were utilised to study thyroid hormone action during early neuronal growth and differentiation. Undifferentiated NT2 cells expressed mRNAs encoding thyroid hormone receptors (TRs) alpha1, alpha2 and beta1, iodothyronine deiodinases types 2 (D2) and 3 (D3) (which act as the pre-receptor regulators), and the thyroid hormone-responsive genes myelin basic protein (MBP) and neuroendocrine specific protein A (NSP-A). When terminally differentiated into post-mitotic neurons (hNT), TRalpha1 and TRbeta1 mRNA expression was decreased by 74% (P=0.05) and 95% (P<0.0001) respectively, while NSP-A mRNA increased 7-fold (P<0.05). However, mRNAs encoding TRalpha2, D2, D3 and MBP did not alter significantly upon neuronal differentiation and neither did activities of D2 and D3. With increasing 3,5,3'-triiodothyronine (T(3)) concentrations, TRbeta1 mRNA expression in cultured NT2 cells increased 2-fold at 10 nM T(3) and 1.3-fold at 100 nM T(3) (P<0.05) compared with that in T(3)-free media but no change was seen with T(3) treatment of hNT cells. D3 mRNA expression in NT2 cells also increased 3-fold at 10 nM T(3) (P=0.01) and 2.4-fold at 100 nM T(3) (P<0.05) compared with control, but there was no change in D3 enzyme activity. In contrast there was a 20% reduction in D3 mRNA expression in hNT cells at 10 nM T(3) (P<0.05) compared with control, with accompanying reductions in D3 activity with increasing T(3) concentrations (P<0.05). There was no significant change in the expression of the TRalpha isoforms, D2, MBP and NSP-A with increasing T(3) concentrations in either NT2 or hNT cells. Undifferentiated NT2 and differentiated hNT cells show differing patterns of T(3)-responsiveness, suggesting that there are different regulatory factors operating within these cell types.

Functionally impaired TR mutants are present in thyroid papillary cancer.

TRs are transcription factors that regulate cell proliferation, differentiation, and apoptosis. They are cellular homologs of the transcriptionally inactive viral oncogene v-erbA. We tested the hypothesis that the functions of TRs could be impaired in cancer tissues as a result of aberrant expression and/or somatic mutations. As a model system, we selected human thyroid papillary cancer, in which the most common abnormalities, RET/papillary thyroid cancer rearrangements (fusion of RET kinase domain to the activating domains of other genes), were found in 40--45% of cases. We found that the mean expression levels of TR beta mRNA and TR alpha mRNA were significantly lower, whereas the protein levels of TR beta 1 and TR alpha 1 were higher in cancer tissues than in healthy thyroid. Sequencing of TR beta 1 and TR alpha 1 cDNAs, cloned from 16 papillary cancers, revealed that mutations affected receptor amino acid sequences in 93.75% and 62.5% of cases, respectively. In contrast, no mutations were found in healthy thyroid controls, and only 11.11% and 22.22% of thyroid adenomas had such TR beta 1 or TR alpha 1 mutations, respectively. The majority of the mutated TRs lost their trans-activation function and exhibited dominant negative activity. These findings suggest a possible role for mutated thyroid hormone receptors in the tumorigenesis of human papillary thyroid carcinoma.

Detection of environmental endocrine‐disruptor effects on gene expression in live Rana catesbeiana tadpoles using a tail fin biopsy technique

We describe a novel method of rapidly assessing exposure of premetamorphic Rana catesbeiana tadpoles to endocrine-disrupting chemicals. Tail fin biopsy and reverse transcription-polymerase chain reaction analyses of the thyroid hormone (TH)-responsive gene, TH receptor beta (TRbeta), demonstrate for the first time that a known accelerant of TH-induced metamorphosis, acetochlor, can significantly enhance TH-induced TRbeta mRNA levels within 24 h at an environmentally relevant dose. Although we focus on laboratory exposures in this study, this method easily can be adapted for use in field studies.

The localization of thyroid hormone receptor mRNAs in human bone.

Thyroid hormones have well-documented effects on the skeleton although the mechanism of their action on bone is poorly understood. We have recently reported the presence of different thyroid hormone receptor isoforms in human bone. However, there is evidence to suggest that the expression of thyroid hormone receptor (TR) protein may not necessarily correlate with its mRNA. In this study, we used specific digoxigenin-labeled ribo probes to investigate the expression of TRalpha1, variant TRalpha2, TRbeta1, and in particular TRbeta2 mRNA in human osteophytic bone and osteoclastoma tissue in situ. The number of positive cells was expressed as the percentage of the total number of cells of the same phenotype. In osteophytes, at sites of endochondral ossification, TRalpha1, variant TRalpha2, TRbeta1, and TRbeta2 mRNA were widely distributed in undifferentiated, proliferating, mature and hypertrophic chondrocytes. At sites of bone remodeling, TRalpha1 mRNA was expressed in the majority (> 90%) of osteoblasts. TRbeta1 and the variant TR-alpha2 mRNA were moderately expressed in approximately 75% of cells with only a few osteoblasts (< 25%) expressing TRbeta2 mRNA. All the TR transcripts were highly expressed in multinucleated osteoclasts in osteoclastoma tissue. The distribution of TR mRNAs was similar to TR receptor protein expression (as we have previously reported) in both osteophytic bone and osteoclastoma tissue except TRalpha1 mRNA that was highly expressed in osteoclasts and in undifferentiated, proliferating, mature, and hypertrophic chondrocytes in contrast to its receptor protein expression. This study highlights the importance of studying both TR mRNA and receptor proteins in triiodothyronine (T3) responsive tissues. This is also the first demonstration of the presence of TRbeta2 mRNA in bone. The role of TRbeta2 in mediating the actions of thyroid hormones in bone is not known and requires further investigation.

Thyroid hormone alpha1 and beta1 receptor mRNA are downregulated by amiodarone in mouse myocardium.

Amiodarone, a powerful antiarrhythmic drug, may exert its effect by antagonism of the thyroid hormone, probably at the receptor level. The aim of this study was to investigate whether amiodarone affects the levels of thyroid hormone receptor (TR) messenger RNA (mRNA) subtypes in mouse hearts. Mice were treated with 10, 25, and 50 mg/kg body weight (BW) amiodarone or vehicle (propyleneglycol) intraperitoneally, daily for 14 days. The heart rate dose-dependently decreased in the 25 mg/kg BW (p < 0.05) and 50 mg/kg BW (p < 0.005) amiodarone-treated mice compared with control. Serum T3 levels were significantly decreased by 25% (4.2 +/- 0.7 pM) in the 50 mg/kg BW amiodarone group in comparison to control (5.6 +/- 1.4 pM; p < 0.05). The serum T4 levels were 1.3 times higher in 50 mg/kg BW amiodarone-treated mice (13.2 +/-1.6 pM) compared with the control (10.3 +/- 1.3 pM; p < 0.005). Determination of TRalpha1, alpha2, beta1, and beta2 mRNA in the heart were performed by reverse transcriptase-polymerase chain reaction (RT-PCR)/enzyme-linked immunosorbent assay (ELISA). Both in treated and untreated mice, TRalpha2 mRNA had the highest density in mouse heart, whereas TRbeta2 mRNA had the lowest density. Amiodarone dose-dependently downregulated the levels of TRalpha1 and beta1 mRNA in comparison to the control. There were, however, no differences in the TRalpha2 and TRbeta2 mRNA levels in the mice heart treated with different doses of amiodarone in comparison with the control group. In conclusion, this study shows that amiodarone subtype selectively downregulates the TR mRNA levels in mouse myocardium in a dose-dependent manner. These results support a thyroid hormone-dependent action of amiodarone.

Role of type III iodothyronine 5-deiodinase gene expression in temporal regulation of Xenopus metamorphosis.

To elucidate the role of type III iodothyronine 5-deiodinase (5-D) in the temporal regulation of amphibian metamorphosis, the regulation of gene expression of 5-D and thyroid hormone receptor beta (TRbeta) in organs of Xenopus laevis was investigated. High levels of TRbeta mRNA in the respective organs were observed at the times of their major morphological changes. Expression of the 5-D gene was highly regulated among the organs during metamorphosis, including up-regulation in the tail and down-regulation in the liver. The tail and liver expressed 5-D gene before their metamorphic changes. These precocious expressions correlated with the lower responsiveness to exogenously added triiodo-L-thyronine (T3) for inducing a high level of TRbeta mRNA expression. However, the same organs responded to lower doses of T3 to regulate 5-D gene expression as seen in spontaneous metamorphosis. The induction of 5-D gene expression was considerably delayed in the intestine, even at an excess dose of T3. Thus, the two genes in a given organ appeared to respond to T3 either with different dose dependencies or with different timetables. The results obtained are also discussed in respect to recent findings in Rana catesbeiana.

Organ specific expression of thyroid hormone receptor mRNA and protein in different human tissues.

The major physiologic effect of thyroid hormone is thought to be initiated by the binding of T3 to the DNA binding thyroid hormone receptor (TR). The aim of this study has been to characterize the organ specific expression of thyroid hormone receptor mRNA, as well as its protein distribution and molecular weight in man. Determination of TRalpha1, alpha2, beta1 and beta2 mRNA molecular size was performed using Northern blot analysis in the human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. TRalpha1, alpha2 and beta1 protein expression was characterized by Western blot analysis of human tissues. TRalpha1 mRNA of 4.9 kb was detected in all 8 tissues analyzed, with varying abundance in the various tissues. TRalpha2 mRNA was detected in 2 different sizes, with higher intensity at 5.7 and lower intensity at 3.2 kb. There were, however, multiple TRbeta1 mRNA of 8, 2 and 1 kb detected. TRbeta1 transcripts of 2 kb and 1 kb showed an organ specific pattern of expression. Multiple TRbeta2 mRNA of 6.6, 5.2, 2.5 and 2.4 kb were detected, as well as a unique 1 kb transcript, in the heart. TRbeta2 transcripts also displayed tissue specific expression. Western blot analysis displayed a single band of 48 kD for TRalpha1. The abundance of the TRalpha1 immunoreactive band was highest in the heart, brain, kidney and skeletal muscle, and lowest in the liver, placenta and lung, while no signals were detected in the spleen. The TRalpha2 specific antibody detected a band of 58 kD in all the tissues analyzed. The relative intensity of the immunoreactive TRalpha2 band detected was highest in the placenta and lung, with a medium concentration range in skeletal muscle, the heart and kidney. The TRalpha2 protein concentration was lowest in the spleen, liver and brain. Human TRbeta1 protein was detected as 55 and 52 kD bands, as well as a unique band of 45 kD in heart. The 52 kD band was detected in all tissues except the kidney and spleen. The 55 kD band was not detected in the brain or liver. Both the 55 and 52 kD TRbeta1 immunoreactive bands were detected in the placenta, lung, heart and skeletal muscle with similar intensity. In conclusion, specific patterns of TR mRNA and protein expression revealed characteristic organ distributions of each subtype. Unique cardiac expression was observed for TRbeta2 mRNA and for TRbeta1 protein.

Prenatal Ethanol Exposure Selectively Reduces the mRNA Encoding α‐1 Thyroid Hormone Receptor in Fetal Rat Brain

Some of the developmental defects characteristic of congenital or experimental hypothyroidism are also observed in children or experimental animals prenatally exposed to ethanol, suggesting that a subset of neurological defects attributable to ethanol exposure are produced by interfering with thyroid hormone action. In this article, we tested whether an ethanol treatment regimen known to produce neurological damage in rats can alter the expression of the mRNAs encoding the thyroid hormone receptor isoforms (TR alpha-1, TR alpha-2, and TR beta-1) in the fetal rat brain neocortex and hippocampus. Rats were fed an ethanol-containing diet beginning on gestational day (G) 6 and continuing until sacrifice on G15, G17, or G21; controls included animals pair-fed a liquid control diet or fed lab chow. Ethanol selectively reduced the expression of TR alpha-1 mRNA in the neocortex and hippocampus on G21, compared with pair-fed and control fetuses. In contrast, pair-feeding selectively reduced TR alpha-2 mRNA in both neocortex and hippocampus on G21, and increased TR beta-1 mRNA on G17. These data support the hypothesis that ethanol may interfere with thyroid hormone action during fetal brain development. In addition, these data indicate that ethanol and pair-feeding exert independent effects on thyroid hormone receptor expression in the developing brain.

Thyroid receptor plasticity in striated muscle types: effects of altered thyroid state.

This study examined nuclear thyroid receptor (TR) maximum binding capacity (Bmax), dissociation constant (Kd), and TR isoform (alpha1, alpha2, beta1) mRNA expression in rodent cardiac, "fast-twitch white," "fast-twitch red," and "slow-twitch red" muscle types as a function of thyroid state. These analyses were performed in the context of slow-twitch type I myosin heavy-chain (MHC) expression, a 3,5,3'-triiodothyronine (T3)-regulated gene that displays varying responsiveness to T3 in the above tissues. Nuclear T3 binding analyses show that the skeletal muscle types express more TRs per unit DNA than cardiac muscle, whereas the latter has a lower Kd than the former. Altered thyroid state had little effect on either cardiac Bmax or Kd, whereas hypothyroidism increased Bmax in the skeletal muscle types without affecting its Kd. Cardiac muscle demonstrated the greatest mRNA signal of TR-beta1 compared with the other muscle types, whereas the TR-alpha1 mRNA signals were more abundant in the skeletal muscle types, especially fast-twitch red. Hyperthyroidism increased the ratio of beta1 to alpha1 and decreased the ratio of alpha2- to alpha1+beta1-mRNA signal across the muscle types, whereas hypothyroidism caused the opposite effects. The nuclear T3 affinity correlated significantly with the TR-beta1 mRNA expression but not with TR-alpha1 mRNA expression. Collectively, these findings suggest that, despite a divergent pattern of TR mRNA expression in the different muscle types, these patterns follow similar qualitative changes under altered thyroid state. Furthermore, TR expression pattern cannot account for the quantitative and qualitative changes in type I MHC expression that occur in the different muscle types.