Cytoplasmic versus nuclear THR alpha expression determines survival of ovarian cancer patients.

PURPOSE: Thyroid hormone receptors (THR) have manifold functions and are involved in the carcinogenesis of several tumor types. Within this study, we aimed to investigate the expression pattern (nuclear versus cytoplasmic) of the THR alpha and its impact on patients survival in ovarian cancer (OvCa). METHODS: The presence of the thyroid hormone receptors THRα, THRα1 and - 2 was investigated in 156 ovarian cancer samples using immunohistochemistry (IHC) using semi-quantitative immunoreactivity (IR) scores and correlated with clinical, pathological data, subtype of ovarian cancer, clinical data, staining of 20 already described OvCa marker proteins and overall survival (OS). RESULTS: Among all subtypes of OvCa, clear cell carcinomas showed the highest THRα expression. Furthermore, nuclear THRα was associated with a reduced survival in this subtype. However, nuclear expressed THRα1 turned out to be a positive prognosticator for all subtypes of OvCa patients. Nuclear THRα2 is a positive prognosticator for OvCa patients of the serous subtype. In contrast, cytoplasmic expression THRα2 was associated with a reduced OS in all subtypes of OvCa patients; while, cytoplasmic expression of THRα1 is associated with reduced OS in mucinous OvCa patients only. In addition, THRα expression correlates with gonadotropin receptors, steroid hormone receptors, TA-MUC1 and glycodelin. CONCLUSION: Depending on nuclear or cytoplasmic expression, our study shows that THRα and its isoforms 1 and 2 provide different prognostic information for ovarian cancer patients. Further investigations should analyze if THRs may represent new endocrine targets for the treatment of ovarian cancer.

Effects of thyroxine on expression of proteins related to thyroid hormone functions (TR-α, TR-ß, RXR and ERK1/2) in uterus during peri-implantation period.

INTRODUCTION: Thyroid hormone is known to play important role during embryo implantation, however mechanisms underlying its actions in uterus during peri-implantation period has not been fully identified. In this study, we hypothesized that thyroid hormone could affect expression of proteins related to its function, where these could explain mechanisms for its action in uterus during this period. METHODS: Female rats, once rendered hypothyroid via oral administration of methimazole (0.03% in drinking water) for twenty-one days were mated with fertile euthyroid male rats at 1:1 ratio. Pregnancy was confirmed by the presence of vaginal plug and this was designated as day-1. Thyroxine (20, 40 and 80 µg/kg/day) was then subcutaneously administered to pregnant, hypothyroid female rats for three days. A day after last injection (day four pregnancy), female rats were sacrificed and expression of thyroid hormone receptors (TR-α and ß), retinoid X receptor (RXR) and extracellular signal-regulated kinase (ERK1/2) in uterus were quantified by Western blotting while their distribution in endometrium was visualized by immunofluorescence. RESULTS: Expression of TRα-1, TRß-1 and ERK1/2 proteins in uterus increased with increasing doses of thyroxine however no changes in RXR expression was observed. These proteins were found in the stroma with their distribution levels were relatively higher following thyroxine treatment. CONCLUSIONS: Increased expression of TRα-1, TRß-1 and ERK1/2 at day 4 pregnancy in thyroxine-treated hypothyroid pregnant rats indicate the importance of thyroxine in up-regulating expression of these proteins that could help mediate the uterine changes prior to embryo implantation.

Thyroid hormone receptor α in breast cancer: prognostic and therapeutic implications.

We determined the expression of two transcriptional variants of thyroid hormone receptor alpha (THRα1 and THRα2) in samples from a cohort of breast cancer patients and correlated expression levels with survival. 130 women who were diagnosed with invasive breast carcinoma between 2007 and 2008 were included. Representative sections of their tumours were analyzed in triplicate on a tissue microarray for expression of THRα1 and THRα2 by immunohistochemistry. The prognostic significance of THRα1 and THRα2 expression was assessed using Kaplan-Meier survival analyses, adjusted for known prognostic factors. Seventy-four percent of tumours had high expression of THRα1 (Allred score ≥6) and 40 % had high expression of THRα2. Expression of THRα2 correlated positively with ER expression (p < 0.001) and with PR expression (p < 0.001), but negatively with HER2 expression (p = 0.018). Patients with low THRα2 expression had inferior 5-year overall survival (75.3 %) compared to those with high expression (91.7 %; p = 0.06). In a multivariate model, high THRα2 expression was a significant and independent prognosticator of improved overall survival (HR = 0.84; 95 % CI 0.71-0.98). Many breast tumours express THRα2 at high levels and these patients experience improved survival. Thyroid hormone signalling may be important in a proportion of breast cancers and THRα2 expression may be a regulator of signalling in this pathway.

Early postnatal exposure to a low dose of decabromodiphenyl ether affects expression of androgen and thyroid hormone receptor-alpha and its splicing variants in mouse Sertoli cells.

Decabromodiphenyl ether (decaBDE) adversely affects reproduction and development. Our previous study showed that postnatal exposure to a low dose of decaBDE (0.025 mg/kg body weight/day) by subcutaneous injection on postnatal days (PNDs) 1 through 5 leads to reductions in testicular size and number of Sertoli cells and sperm, while higher dose of decaBDE (2.5 mg/kg body weight/day) had no significant differences about these. In the present study, we examined the molecular mechanism of these effects on mouse testes following postnatal exposure to a low decaBDE dose. We hypothesized that postnatal exposure to decaBDE may alter levels of serum thyroid hormones (THs) and testosterone, or the level of TH receptor alpha (Thra) transcripts and its splicing variants and androgen receptor (Ar) in Sertoli cells, adversely affecting spermatogenesis. To test this hypothesis, we examined serum TH and testosterone levels and the levels of transcripts of the Ar, Thra and its splicing variants, and Thra splicing factors (Hnrnpa1, Srsf1, and Hnrnph1) with qPCR in isolated mouse Sertoli cells exposed postnatally to decaBDE (0.025, 0.25, and 2.5 mg/kg). Levels of serum testosterone and transcripts encoding Ar, Thra, and its variant, Thra1, declined significantly in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg. No significant differences in serum TH level or Thra2, Hnrnph1, or Srsf1 transcript levels were observed between control and decaBDE-exposed mice. However, the Thra1:Thra2 and Hnrnpa1:Srsf1 ratios were altered in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg but not in cells exposed to 0.25 or 2.5 mg decaBDE/kg. These results indicate that postnatal exposure to a low dose of decaBDE on PNDs 1 through 5 lowers the testosterone level and the levels of Ar and Thra transcripts in Sertoli cells, accompanied by an imbalance in the ratios of Thra splicing variants, resulting in smaller testicular size and impaired spermatogenesis.

Maternal thyroid hormones are essential for neural development in zebrafish.

Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.

The pattern of expression and role of triiodothyronine (T3) receptors and type I 5'-deiodinase in breast carcinomas, benign breast diseases, lactational change, and normal breast epithelium.

AIM: : To study the pattern of expression of triiodothyronine (T3) receptors and type I 5'-deiodinase in various breast pathologies comparing malignant and nonmalignant epithelia that include lactational change. METHODS AND RESULTS: A retrospective study was performed on formalin-fixed, paraffin-embedded archival material from 146 cases of carcinomas, normal breast tissue, breast tissue showing lactational change, and benign breast lesions. Archive tissue blocks were selected and sections were cut for immunohistochemistry to study the expression of thyroid hormone receptor α-1 (THR-α1) in the cytoplasm and nuclei of cells in tissues under study. Thick sections were cut for type I 5'-deiodinase evaluation using reverse transcriptional PCR.THR-α1 showed no nuclear expression in the carcinoma group. Combined nuclear and cytoplasmic expression was seen in 47.6%, 63.4%, 64.3%, and 58.3% in the benign, fibrocystic, fibroadenoma, and lactational change groups, respectively, compared with only 17.4% of cases in the carcinoma group. This suggests deregulation of the thyroid hormone in breast cancer. Theories for the possible role of thyroid hormone in the pathogenesis of breast cancer are discussed.Type I 5'-deiodinase was not shown to be differentially expressed in malignant versus nonmalignant groups. CONCLUSIONS: Our study revealed substantial reduction in the protein expression profile of THRs in malignant versus nonmalignant mammary epithelium suggesting a possible role in breast cancer development. The presence of THRs in mammary epithelium seems to be protective against the development of breast cancer. This could serve as a potential prognostic and therapeutic target for breast cancer.

Leptin administration restores the fasting-induced increase of hepatic type 3 deiodinase expression in mice.

BACKGROUND: Decreased serum leptin has been proposed as a critical signal initiating the neuroendocrine response to fasting. Leptin administration partially reverses the fasting-induced suppression of the hypothalamus-pituitary-thyroid axis at the central level. It is, however, unknown to what extent leptin affects peripheral thyroid hormone metabolism. The aim of this study was to evaluate the effect of leptin administration on starvation-induced alterations of peripheral thyroid hormone metabolism in mice. METHODS: Three types of experiments were performed: (i) mice were fasted for 24 hours while leptin was administered twice (at 0 and 8 hours, 1 µg/g body weight [BW]), (ii) mice were fasted for 24 hours and, subsequently, leptin was given once at 24 hours (killed at 28 and 32 hours), and (iii) mice were fasted for 48 hours. All groups had appropriate controls. Serum triiodothyronine and thyroxine, liver type 1 deiodinase (D1), type 3 deiodinase (D3), thyroid hormone receptor (TR)ß1, TRα1 and α2 mRNA expression, and liver D1 and D3 activity were measured. RESULTS: Twenty-four hours of fasting decreased liver TRß1 mRNA expression, while liver TRα1, TRα2, and D1 mRNA expression and activity did not change. In contrast, 24 hours of fasting increased liver D3 mRNA. Leptin administration after fasting restored liver D3 expression, while serum thyroid hormone levels and liver TRß1 expression remained low. CONCLUSION: Leptin administration selectively restores starvation-induced increased hepatic D3 expression independently of serum thyroid hormone concentrations. The present study shows that fasting-induced changes in mRNA expression of genes involved in hepatic hormone metabolism are influenced not only by decreased serum thyroid hormone levels but also by serum leptin.

Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock.

AIM: Non-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo. METHODS: Serum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators. RESULTS: Patients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRß (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48  h. CONCLUSIONS: Patients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

Severe food restriction induces myocardial dysfunction related to SERCA2 activity.

Previous studies have shown that food restriction promotes myocardial dysfunction in rats. However, the molecular mechanisms that are responsible are unclear. We investigated the role of sarcoplasmic reticulum Ca2+-ATPase (SERCA2) on myocardial performance in food-restricted rats. Male Wistar-Kyoto rats, 60 days old, were fed a control or restricted diet (daily energy intake reduced to 50% of the control) for 90 days. Expression of Serca2a, phospholamban (PLB), Na+/Ca2+ exchanger (NCX), and thyroid hormone receptor (TRalpha1, TRbeta1) mRNA was determined by quantitative PCR. SERCA2 activity was measured by using 20 micromol/L cyclopiazonic acid (CPA) in a left ventricular papillary muscle preparation during isometric contraction in basal conditions and during post-rest contraction. Serum concentrations of thyroxine (T4) and thyrotropin (TSH) were also determined. The 50%-restricted diet reduced body and ventricular weight and serum T4 and TSH levels. The interaction of CPA and food restriction reduced peak developed tension and maximum rate of tension decline (-dT/dt), but increased the resting tension intensity response during post-rest contraction. PLB and NCX mRNA were upregulated and TRalpha1 mRNA was downregulated by food restriction. These results suggest that food restriction promotes myocardial dysfunction related to impairment of sarcoplasmic reticulum Ca2+ uptake as a result of a hypothyroid state.

[Prokaryotic expression and polyclonal antibody preparation of TRalphaA in Japanese flounder Paralichthys olivaceus].

To study the role of the thyroid hormone receptor TRalphaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRalphaA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl beta-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1:200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRalphaA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRalphaA in vivo.

The expression of thyroid hormone receptor isoforms in human astrocytomas.

BACKGROUND: Thyroid hormone plays a major role in normal mammalian brain maturation and affects the development of astrocytes. The expression of TR isoforms has been studied in different neoplasias. Increasing evidence has suggested that aberrant expression of TR isoforms could be associated with tumorigenesis. However, little was studied about the expression of TR isoforms in human astrocytomas. METHODS: In this study, RT-PCR was used to examine the expression of human TR isoforms in 34 human astrocytoma samples. RESULTS: We compared the TR expression between low grade (WHO grade II) and high grade (WHO grade III and IV). The frequency of TRalpha1 or TRalpha2 expression significantly decreased with the grade of malignancy (P=.005 and P=.043, respectively). However, the frequency of TRbeta1 expression significantly increased with the grades of malignancy astrocytomas (P=.017). CONCLUSIONS: Our study demonstrated for the first time that TR isoforms are indeed expressed in human astrocytomas. The expression of TR isoforms is correlated to the malignancy grading of astrocytomas. Our result provides insight into the potential use of hormonal therapy for brain tumors that overexpress or underexpress TRs.

Thyroid hormone receptor alpha 1: a switch to cardiac cell "metamorphosis"?

Thyroid hormone receptor alpha1 (TRalpha1) is predominantly expressed in the myocardium but its biological function under physiological or pathological conditions remains largely unknown. The present study investigated possible interactions between alpha1 adrenergic and thyroid hormone signaling at the level of TRalpha1, potential underlying mechanisms and physiological consequences, as well as the role of TRalpha1 in cell differentiation. This may be of physiological relevance since both thyroid hormone and adrenergic signalling are implicated in the pathophysiology of cardiac remodelling. Neonatal cardiomyocytes obtained from newborn rats (2-3 days) were exposed to phenylephrine (PE, an alpha1 adrenergic agonist) for 5 days, in the absence or excess of T3 in the culture medium. PE, in the absence of T3, resulted in 5.0 fold increase in TRalpha1 expression in nucleus and 2.0 fold decrease in TRalpha1 expression in cytosol, P<0.05. As a result, a fetal pattern of myosin isoform expression with marked expression of beta-MHC was observed in PE treated vs the untreated cells, P<0.05. PD98059 (an ERK signalling inhibitor) abrogated this response. In the presence of T3 in the culture medium, TRalpha1 expression was increased 1.6 fold in nucleus and 2.0 fold in cytosol in PE-T3 vs PE treated cells, P<0.05, and the fetal pattern of myosin isoform expression was prevented. Parallel studies with H9c2 myoblasts showed that reduction of T3 binding to TRalpha1 receptor delayed cardiac myoblasts differentiation without affecting proliferation. In conclusion, in neonatal cardiomyocytes, nuclear TRalpha1 is overexpressed after prolonged activation of the alpha1- adrenergic signalling by PE. This response seems to be an ERK kinase dependent process. Over-expression of TRalpha1 may lead to fetal cardiac phenotype in the absence of thyroid hormone availability. Furthermore, TRalpha1 seems to be critical in cardiac myoblast differentiation.

Age-related changes in renal and hepatic cellular mechanisms associated with variations in rat serum thyroid hormone levels.

Aging is associated with changes in thyroid gland physiology. Age-related changes in the contribution of peripheral tissues to thyroid hormone serum levels have yet to be systematically assessed. Here, we investigated age-related alterations in the contributions of the liver and kidney to thyroid hormone homeostasis using 6-, 12-, and 24-mo-old male Wistar rats. A significant and progressive decline in plasma thyroxine occurred with age, but triiodothyronine (T(3)) was decreased only at 24 mo. This was associated with an unchanged protein level of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in the kidney and with a decreased MCT8 level in the liver at 24 mo. Hepatic type I deiodinase (D1) protein level and activity declined progressively with age. Renal D1 levels were decreased at both 12 and 24 mo but D1 activity was decreased only at 24 mo. In the liver, no changes occurred in thyroid hormone receptor (TR) TRalpha(1), whereas a progressive increase in TRbeta(1) occurred at both mRNA and total protein levels. In the kidney, both TRalpha(1) and TRbeta(1) mRNA and total protein levels were unchanged between 6 and 12 mo but increased at 24 mo. Interestingly, nuclear TRbeta1 levels were decreased in both liver and kidney at 12 and 24 mo, whereas nuclear TRalpha(1) levels were unchanged. Collectively, our data show differential age-related changes among hepatic and renal MCT8 and D1 and TR expressions, and they suggest that renal D1 activity is maintained with age to compensate for the decrease in hepatic T(3) production.

Locomotor deficiencies and aberrant development of subtype-specific GABAergic interneurons caused by an unliganded thyroid hormone receptor alpha1.

Thyroid hormone (TH) deficiency during development causes severe and permanent neuronal damage, but the primary insult at the tissue level has remained unsolved. We have defined locomotor deficiencies in mice caused by a mutant thyroid hormone receptor alpha1 (TR alpha1) with potent aporeceptor activity attributable to reduced affinity to TH. This allowed identification of distinct functions that required either maternal supply of TH during early embryonic development or sufficient innate levels of hormone during late fetal development. In both instances, continued exposure to high levels of TH after birth and throughout life was needed. The hormonal dependencies correlated with severely delayed appearance of parvalbumin-immunoreactive GABAergic interneurons and increased numbers of calretinin-immunoreactive cells in the neocortex. This resulted in reduced numbers of fast spiking interneurons and defects in cortical network activity. The identification of locomotor deficiencies caused by insufficient supply of TH during fetal/perinatal development and their correlation with subtype-specific interneurons suggest a previously unknown basis for the neuronal consequences of endemic cretinism and untreated congenital hypothyroidism, and specifies TR alpha1 as the receptor isoform mediating these effects.

Reduced expression of thyroid hormone receptors and beta-adrenergic receptors in human failing cardiomyocytes.

An altered thyroid hormone profile has been reported in patients with congestive heart failure. However, information regarding the status of thyroid hormone receptors in human failing cardiomyocytes is lacking. Therefore the expression of thyroid hormone and beta-adrenergic receptors was investigated in human ventricular cardiomyocytes isolated from patients with end-stage heart failure (FM, n=12), or from tentative donors (C, n=4). The expression of thyroid (TRalpha1, and TRbeta1) and beta-adrenergic receptors (ARB1 and ARB2) was measured at both the gene, and at the protein level. In FM the reduced mRNA expression of ARB1 (p<0.05, -37%) and ARB2 (p<0.05, -42%) was associated with a reduction of the messenger for TRalpha1 (p<0.05, -85%) and TRalpha2 (p<0.05, -73%). These findings were confirmed at the protein level for ARB1, ARB2 and TRalpha1. These data reveal that in human heart failure the reduction of beta-adrenergic receptors is associated with reduced expression of both TRalpha1 and TRalpha2 isoforms of thyroid hormone receptors.

Overexpression of E2F1 in clear cell renal cell carcinoma: a potential impact of erroneous regulation by thyroid hormone nuclear receptors.

We show here that the promoter of E2F1 gene, encoding one of the key regulators of cell proliferation, is overly active in the presence of low amounts of triiodothyronine (T3) and in the presence of mutant thyroid hormone receptor. We also show that T3-thyroid hormone receptor pathway of regulation of molecular processes is disturbed in clear cell renal cell carcinoma (ccRCC) on several levels, including overexpression of thyroid hormone receptors and the disturbance of their binding to DNA and to the hormone. In comparison to the cancer-free kidneys and peritumoral respective control tissues, E2F1 mRNA and protein levels are significantly increased in cancer tissues. A significant correlation between E2F1 mRNA and protein levels has been found in both control types and ccRCCs. No correlation was observed between the amount of E2F1 mRNA and the amount of thyroid hormone receptors or their DNA or T3 binding activity, suggesting that the function of thyroid hormone receptors could be markedly disturbed in both tumor and peritumoral cells. In summary, we show that ccRCC is characterized by the overexpression of E2F1, which is likely a result of a deregulated control of T3-dependent molecular processes.

Thyroid hormone receptor and IGF1/IGFR systems: possible relations in the human heart.

Thyroid hormone (TH) and insulin growth factor 1 (IGF1) systems both play crucial roles in the regulation of cardiac remodeling and hypertrophy processes. The mediation of this regulation is attributed to specific thyroid hormone receptors (TRs) and to the IGF1 receptor (IGF1R). In humans, two TR genes are expressed in the heart, TRalpha and TRbeta. Each gene generates two isoforms: TRalpha1, TRalpha2 and TRbeta1, TRbeta2. The aim of the present work was to study the local thyroid hormone and IGF1 signaling in human myocardium through the evaluation of the gene expression of TRalpha1, TRalpha2, TRbeta1 and IGF1R among atrial and ventricular biopsies obtained from patients undergoing cardiac surgery. Moreover, we evaluated possible correlations between TR and IGF1/IGF1R systems. Eighteen clinically and biochemically euthyroid patients (aged 68.3+/-3.2years, mean+/-SEM) without overt heart failure (Ejection Fraction (EF), 46.4+/-2.8%; Left Ventricular End Diastolic Diameter (LVEDD), 54.3+/-1.2mm, mean+/-SEM; NYHA I-II) were enrolled in the study: 13 undergoing aorto-coronary bypass and 5 undergoing valve replacement (aortic/mitral valve). The examination of total RNA, using real time PCR (LightCycler Technology) confirmed the expression of specific mRNAs encoding TRalpha1, TRalpha2, TRbeta1 and both IGF1 and IGF1R. We found that the three TR genes are co-expressed in the human atrium and ventricle. The finding of a strong correlation among IGF1R and the three TR genes expressed in the atrium (p<0.001) and among the three TRs in the atrium (p<0.001) suggests the interesting possibility that the two systems, TRs and IGF1R could also be functionally associated.

Effects of induced systemic hypothyroidism upon the retina: regulation of thyroid hormone receptor alpha and photoreceptor production.

PURPOSE: Investigate the effects of systemic hypothyroidism upon the differentiated, growing, and regenerating retina of postmetamorphic winter flounder, a vertebrate that experiences a thyroid hormone (TH) induced metamorphosis during development. METHODS: A loss-of-signal strategy was utilized in which TH signaling was disrupted by inhibiting TH synthesis. Induced hypothyroidism was confirmed by radioimmunoassay. Reverse transcriptase PCR (RT-PCR), real-time quantitative PCR (qPCR), molecular cloning, non-isotopic in situ hybridization, western blot analysis, and indirect immunohistochemistry techniques were performed to analyze retinal thyroid hormone receptors (TR), photoreceptor production, and the phenotypic repertoire of differentiated retinal cells as a function of TH signaling status. RESULTS: Molecular bases for TH signaling were supported by retinal expression of TH receptors alpha and beta. TH-dependent transcriptional regulation of TRalpha but not TRbeta was indicated, with induced hypothyroidism producing an increase in TRalpha expression. Evidence for post-transcriptional regulation of retinal TRalpha was observed. The repertoire of inner retinal cell types in premetamorphic fish (a naturally low TH condition) matched that observed in the central retinas of both normal postmetamorphic fish (a naturally elevated TH condition) and postmetamorphic fish rendered hypothyroidic. In differentiated postmetamorphic retina there was no evidence for significant differences in opsin expression between normal and hypothyroidic animals. Induced hypothyroidism did, however, significantly affect the types of photoreceptors that were produced in postmetamorphic retina: as a hypothyroidic postmetamorphic retina grew or regenerated following injury, the phenotypic repertoire of newly-produced photoreceptors matched that observed for premetamorphic retina, in which rods, SWS2-expressing "blue" cones, and LWS-expressing "red" cones are absent, and only the RH2-expressing "green" cone type is present. The effects of induced hypothyroidism upon photoreceptor specification (manifestation of the rod lineage) and differentiation (expression of a particular opsin by specified cones) were apparently reversible. CONCLUSIONS: The results suggest a TH-dependent regulation of retinal TRalpha, a lack of TH-dependent regulation of the phenotypic identity of differentiated retinal cells, and the operation of similar cytogenic mechanisms during retinal growth and regeneration. The principal conclusion is that TH signaling significantly affects, in a targeted manner, the production of both rod and cone photoreceptors during retinal growth and regeneration.

Effects of bisphenol A on thyroid hormone-dependent up-regulation of thyroid hormone receptor alpha and beta and down-regulation of retinoid X receptor gamma in Xenopus tail culture.

We investigated effects of different concentrations (10(-7) - 10(-5) M) of bisphenol A (BPA), which is known as an estrogenic and anti-thyroid hormonal endocrine disrupter, on the expression of thyroid hormone receptor (TR) alpha and beta and retinoid X receptor (RXR) gamma mRNA in tails of stage 52-54 Xenopus tadpoles in organ culture in the presence or absence of different concentrations of triiodo-thyronine (T(3)). In the absence of T(3), BPA at any concentration examined did not show remarkable effects on tail length but blocked 10(-7) M T(3)-induced tail resorption in a concentration-dependent manner. Semi-quantitative analyses of TRalpha and TRbeta mRNAs by RT-PCR in the tail specimens indicated that BPA shows an apparent antagonistic effect towards the receptors and reduced their mRNA levels relative to controls. When administered together with 10(-7) M T(3), the antagonistic effects of BPA were detected more clearly and dose-dependently. While BPA prevented the autoinduction of both TRalpha and TRbeta genes by T(3), the effect was less marked on TRalpha than on TRbeta. BPA also moderately suppressed RXRgamma gene expression. Gene expression of RXRgamma, a partner for heterodimer formation of TRs, was supressed by T(3) alone and also by BPA alone, but no additive effects were observed so far as studied. The present study indicates that a relatively low concentration of BPA, 10(-7) M, as compared with those examined previously (10(-5) to 10(-4) M) by us and other investigators, acts as an antagonist of T(3) through suppression of TRalpha and TRbeta gene expression in Xenopus tail in culture.

Molecular characterization of thyroid hormone receptors from the leopard gecko, and their differential expression in the skin.

Thyroid hormones (THs) play crucial roles in various developmental and physiological processes in vertebrates, including squamate reptiles. The effect of THs on shedding frequency is interesting in Squamata, since the effects on lizards are quite the reverse of those in snakes: injection of thyroxine increases shedding frequency in lizards, but decreases it in snakes. However, the mechanism underlying this differential effect remains unclear. To facilitate the investigation of the molecular mechanism of the physiological functions of THs in Squamata, their two specific receptor (TRalpha and beta) cDNAs, which are members of the nuclear hormone receptor superfamily, were cloned from a lizard, the leopard gecko, Eublepharis macularius. This is the first molecular cloning of thyroid hormone receptors (TRs) from reptiles. The deduced amino acid sequences showed high identity with those of other species, especially in the C and E/F domains, which are characteristic domains in nuclear hormone receptors. Expression analysis revealed that TRs were widely expressed in many tissues and organs, as in other animals. To analyze their role in the skin, temporal expression analysis was performed by RT-PCR, revealing that the two TRs had opposing expression patterns: TRalpha was expressed more strongly after than before skin shedding, whereas TRbeta was expressed more strongly before than after skin shedding. This provides good evidence that THs play important roles in the skin, and that the roles of their two receptor isoforms are distinct from each other.