TRbeta Expression Research Articles

Thyroid hormone receptor (TR)alpha and TRbeta expression in breast cancer.

There is evidence that breast cancer patients suffer from thyroid disorders. However, the relation between thyroid receptor (TR) expression and breast cancer remains unknown so far. Therefore, the aim of this study was an immunohistochemical analysis of TR expression in breast cancer patients. The expression of the combined antibody TRalpha1 and 2 and TRalpha1 or 2 alone as well as the expression of combined TRbeta1 and 2 and TRbeta1 or 2 alone was investigated with specific monoclonal or polyclonal antibodies in 82 patients. All patients presented with a first diagnosis of sporadic breast cancer. The ABC method was used for staining and staining intensities were analyzed using the IRS score. Both TRalpha and TRbeta were expressed in the nuclei of breast cancer cells. In 24% (28/78) of the slides TRalpha1 and 2 IRS was positive. Immunopositivity for TRalpha1 was found in 55/78 slides, for TRalpha 2 in 54/79 slides (71 and 68%, respectively). The expression of TRbeta1 and 2 showed a positive detection in 33/77 (43%) of the slides, for TRbeta1 it was 43/79 (54%), for TRbeta2 60/76 (79%). Significant correlations of the expression of TRs - especially TRalpha2 - were found with further prognostic histopathological parameters such as tumor size, axillary lymph node involvement, grading and hormone receptor status. Multivariate analysis showed a trend for TRalpha2 as an independent predictor of disease-free and overall survival. Our results revealed specific alterations in the expression of TRs - especially of TRalpha2 - in breast cancer patients, suggesting it as a marker with possible prognostic validity.

IMPACT OF EXPOSURE TO COMPLEX CHEMICAL MIXES FOUND IN WASTEWATER EFFLUENT ON THYROID HORMONE-ORGANIZED DEVELOPMENT

Event Abstract Back to Event IMPACT OF EXPOSURE TO COMPLEX CHEMICAL MIXES FOUND IN WASTEWATER EFFLUENT ON THYROID HORMONE-ORGANIZED DEVELOPMENT Catherine Propper1*, Brian Searcy1, Angela Schwendiman1, Claire Ramirez1, Michael Owen1, Nik Veldhoen2 and Caren Helbing2 1 Northern Arizona University, Biological Sciences, United States 2 University of Victoria, Biochemistry and Microbiology, Canada Anthropogenic chemicals impact development, reproduction and behavior in wildlife and laboratory animals. Organisms living downstream from wastewater effluent (WWE) outlets are exposed to complex mixes of such compounds, and some exposed organisms show disrupted developmental processes. Using Xenopus laevis as a model, we tested the hypothesis that exposure to WWE decreased time to metamorphosis. Tadpoles were exposed from NF-stage 47 through the completion of metamorphosis to either artificial pond water, RO water balanced to equal salts in local WWE, or WWE. Animals exposed to WWE underwent metamorphosis sooner than either control group. Since thyroid hormone directly controls metamorphosis, our results suggest the WWE had thyroid hormone-like activity. To test this hypothesis, we used X. laevis whole tail cultures to determine whether WWE was impacting expression of four genes sensitive to thyroid hormone signaling: thyroid hormone receptor beta (TRbeta), deiodinase II (D2), corticotropin releasing hormone binding protein (CRHBP), and fibroblast activation protein alpha (FAPalpha). Tails were incubated for 48 hr to 0 or 10nM thyroid hormone (T3) and either culture media alone or extracts of WWE reconstituted in culture media to 1X or 5X the original WWE concentration. T3 induced a significant increase in mRNA in TRbeta, CRHBP, and FAPalpha, but not D2. Wastewater effluent alone induced a significant increase in FAPalpha (at both 1X and 5X) and CRHBP (at 1X only) expression, but not expression of TRbeta or D2. When tails were exposed to both T3 and WWE, all genes showed large, significant synergistic increases in expression compared to tails exposed to T3 or WWE alone. Our results suggest the decreased time to metamorphosis found in vivo is not solely a direct result of WWE compounds binding to TH receptor, but that some of the compounds have the potential to affect thyroid hormone signaling pathways upstream and/or downstream of receptor activation. Acknowledgements Acknowledgement: This work was supported by Arizona State TRIF funds to CRP. Keywords: Effluent, endocrine disruption, Gene Expression, Thyroid hormone, wastewater, Xenopus Conference: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology, Ann Arbor, United States, 11 Jul - 13 Jul, 2011. Presentation Type: Invited Symposium Topic: Endocrine disruption Citation: Propper C, Searcy B, Schwendiman A, Ramirez C, Owen M, Veldhoen N and Helbing C (2011). IMPACT OF EXPOSURE TO COMPLEX CHEMICAL MIXES FOUND IN WASTEWATER EFFLUENT ON THYROID HORMONE-ORGANIZED DEVELOPMENT. Front. Endocrinol. Conference Abstract: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology. doi: 10.3389/conf.fendo.2011.03.00038 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Catherine Propper, Northern Arizona University, Biological Sciences, Flagstaff, AZ, 86011, United States, Catherine.Propper@nau.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing Google Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing Google Scholar Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing PubMed Catherine Propper Brian Searcy Angela Schwendiman Claire Ramirez Michael Owen Nik Veldhoen Caren Helbing Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Triclosan and Anuran Metamorphosis: No Effect on Thyroid-Mediated Metamorphosis in Xenopus laevis

Nieuwkoop and Faber stage 51 Xenopus laevis larvae were exposed for 21 days to four different concentrations of triclosan (TCS): <0.2 (control), 0.6, 1.5, 7.2, or 32.3 microg TCS/l. Primary endpoints were survival, hind limb length, body length (whole; snout to vent), developmental stage, wet whole body weight, and thyroid histology. Thyroid hormone (TH) concentrations were determined in whole thyroid and plasma samples from stage-matched exposure day 21 specimens. TH receptor-beta (TRbeta) expression was measured in stage-matched tail fin tissue samples collected at exposure days 0 and 21. Reduced larval growth occurred at exposure day 21 with 1.5 microg/l treatment. Larval developmental stage at exposure day 21 was not significantly different from controls based on observed parameters. Thyroid histology was not affected by TCS, and thyroxine (T4) levels in thyroid glands or plasma were not different from controls. A concentration-dependent increase in TRbeta expression in exposure day 21 larvae was not detected. However, increased expression was found in stage-matched larvae exposed to 1.5 or 7.2 microg TCS/l. Our study indicates that environmentally relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model.

Teratogenic Effects of Chronic Treatment with Corticosterone on Tadpoles of Xenopus laevis

The present study investigated effects of chronic treatment with corticosterone (B) on larval development of the amphibian Xenopus laevis. Tadpoles (stage 51) were treated with B (100, 500 nmol/L), 50 mg/L of the anti-thyroidal substance ethylenethiourea (ETU), and a combination of ETU (50 mg/L) plus B (500 nmol/L) to address corticosteroid effects in the presence or absence of thyroid hormones (TH). ETU-treated tadpoles displayed developmental arrest, reduced levels of TH receptor beta (TRbeta) and prolactin (PRL) mRNAs, but increased expression of thyroid-stimulating hormone (TSHbeta). Tadpoles exposed to B were characterized by asynchrony of metamorphic tissue remodeling and decreased mRNA expression of corticotropin-releasing factor (CRF). Cotreatment of ETU and B reversed the effects of ETU on PRL and TSHbeta expression but did not alter ETU effects on development and TRbeta expression. Our findings confirm that chronic treatment with high doses of corticosteroids has teratogenic effects on developing tadpoles and additionally suggest a stimulatory effect of B on gene expression of the antimetamorphic pituitary hormone PRL.

Transient expression of thyroid hormone nuclear receptor TRβ2 sets S opsin patterning during cone photoreceptor genesis

Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.

The persistent DDT metabolite, 1,1‐dichloro‐2,2‐bis(p‐chlorophenyl)ethylene, alters thyroid hormone‐dependent genes, hepatic cytochrome P4503A, and pregnane × receptor gene expressions in atlantic salmon (Salmo salar) parr

The present study investigated the effects of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) on the thyroid and steroid-metabolizing system in Atlantic salmon (Salmo salar) parr. Fish were exposed to waterborne DDE and thyroxine (T4), both singly and in combination, for 5 d. Thyroid-stimulating hormone (TSHbeta), T4 deiodinase (T4ORD), thyroid receptors (TRalpha and TRbeta), and insulin-like growth factor type 1 receptor (IGF-1R) were analyzed using quantitative (real-time) polymerase chain reaction in liver, brain, and kidney, whereas cytochrome P4503A (CYP3A) and pregnane X receptor (PXR) mRNA levels were analyzed only in the liver. Exposure to DDE and T4, both singly and in combination, inhibited TSHbeta expression in the brain. The DDE induced TSHbeta in the liver, and T4 inhibited TSHbeta in the liver and kidney, both singly and in combination with DDE. The DDT-metabolite DDE induced T4ORD expression in the kidney and liver, and combined exposure with T4 inhibited T4ORD expression in the brain, kidney, and liver. The IGF-1R and TRalpha expressions were induced by DDE and T4 singly in the brain, whereas combined exposure with both compounds did not affect IGF-1R and TRd transcript levels. Whereas T4 inhibited TRbeta expression in the liver, exposure to DDE, both singly and in combination with T4, induced TRbeta transcript levels in the liver. Exposure to T4 and DDE, both singly and in combination, resulted in a parallel pattern of CYP3A and PXR mRNA induction in the liver. These results indicate that DDE alters thyroid hormone-dependent genes and hepatic CYP3A and PXR levels. The hepatic modulation of CYP3A and PXR transcript levels by DDE represents a novel aspect of DDE toxicity that, to our knowledge, has not been demonstrated previously in fish. Therefore, the present study demonstrates some possible physiological and endocrine consequences from exposure to endocrine-disrupting chemicals for salmon parr during smoltification.

Expression of thyroid hormone receptor/erbA genes is altered in human breast cancer.

The relation between thyroid status and diseases and cancer is unclear. No detailed analysis of thyroid hormone receptor (TR) expression in human breast cancer has been reported. We have analysed the expression and mutational status of the TRalpha1, encoded by the c-erbA proto-oncogene, TRbeta1 and TRbeta2 isoforms in 70 sporadic breast cancers. Alterations in the RNA level of TRbeta1, TRalpha1, or both were found in a number of patients. No expression of TRbeta2 RNA was detected. Western blotting analysis confirmed the differences in expression at the protein level in those cases where sufficient tumor sample was available. Additionally, tumor-specific truncated TRbeta1 RNA was found in six patients. Strikingly, three transcripts shared the same breakpoint. Only one tumor carried the corresponding deletion at the genomic DNA level, suggesting that the remaining abnormal TRbeta1 transcripts are aberrant splicing products. Though no significant correlation was found between TRbeta1 alteration and any clinical parameter, it showed a tendency to associate with early age of onset (<50 years). Our results reveal specific alterations in the expression of TRbeta and TRalpha genes in a subset of breast cancer patients, suggesting that deregulation of thyroid hormone target genes may be involved in the generation of this neoplasia.

Ontogenetic pattern of thyroid hormone receptor expression in the human testis.

We studied the spatiotemporal distribution of thyroid hormone nuclear receptors (TRs) alpha1 and alpha2 and beta messenger RNA (mRNA) levels in normal human testicular tissue during development and in adulthood. Nonpathological specimens from five aborted fetuses (17 and 23 weeks of gestation, three and two cases, respectively) and from four patients undergoing orchiectomy (18 months old and 38-, 42-, and 52-yr-old, respectively) were analyzed by Northern blot, semiquantitative RT-PCR amplification using DNA sequences or specifically designed primers for the TR isoforms, and in situ hybridization. By using PCR amplification, we found that TRalpha1 and TRalpha2 are both expressed at different levels in fetal and adult testis. At all ages TRalpha2 is found at higher levels. Northern analysis showed hybridization signals corresponding to the expression of TRalpha2 and TRalpha in a ratio that increased from 2.6 at 17 weeks of gestation to 12.0 in adulthood. In fact, the expression of TRalpha1 dramatically decreased throughout development, being faintly detectable in the adult testis. Expression of TRbeta was not detected at any age studied. This finding was further confirmed by PCR, which did not amplify TRbeta either in fetal or in adult testis mRNAs. In situ hybridization studies showed the absence of TRbeta and that TRalpha1 and TRalpha2 colocalized in Sertoli cells of prepubertal testis, whereas germ and interstitial cells appeared devoid of TR mRNA signals. From these results it can be concluded that the human testis exclusively expresses TRalpha, which is localized in Sertoli cells, TRbeta being always undetectable. Fetal and prepubertal ages represent the period of maximal expression of TRalpha1 and TRalpha2. The alpha2/alpha1 ratio rises dramatically after development. These results confirm a critical window for the action of thyroid hormone in human testis, in the period of maximal expression of T3 binding isoform TRalpha1, and may account for the macroorchidism without virilization occurring when hyposecretion of thyroid hormones occurs before puberty.

Thyroid hormone receptor genes of neotenic amphibians.

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to alpha and beta genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3',5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRalpha in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRbeta expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRalpha and TRbeta may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis.

Importance of the most proximal GC box for activity of the promoter of human thyroid hormone receptor beta 1.

We wish to localize the sequences required for transcriptional expression of the thyroid hormone receptor beta 1 (TR beta 1). Constitutive activity of the promoter of human thyroid hormone receptor beta 1 was assessed by transient transfection of deletion constructs attached to luciferase as reporter, into P19, GH3, HepG2, H19-7, and COS1 cells. A 40-base pair fragment of the beta 1 promoter including the TATA box induced minimal luciferase activity, which was considered basal activity. The activities of various lengths of the beta 1 promoter were estimated relative to the minimal promoter in five cell lines. The region between -130 and -40 was crucial for constitutive activity in all cell lines. Further deletion analysis in HepG2 cells showed that two regions mainly augmented the transcriptional activity of the minimal 40 base pair fragment. One region located at -115 to -93, which is highly GC-rich, included the most proximal of five putative GC boxes present in the whole 1325-base pair promoter. A second region contributing to expression of TR beta 1 in HepG2 cells is at -70 to -40. Mutation of the most proximal GC box strongly suppressed transactivity of the whole promoter in P19 and HepG2 cells. In contrast, mutations in the other GC boxes did not suppress transactivation in P19 cells and slightly suppress activation in HepG2 cells. In Schneider cells, which do not express Sp1, transactivity of the region distal to -40 is positively regulated by cotransfection with a vector expressing Sp1.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Distribution of the Endogenous and Transfected β Form of Thyroid Hormone Nuclear Receptor Visualized by the Use of Domain-Specific Monoclonal Antibodies

To study the regulation, tissue distribution, and subcellular localization of nuclear receptor for thyroid hormone, monoclonal antibodies (mAbs) against the human placental c-erbA (hTR beta 1) protein were prepared. hTR beta 1 was expressed in Escherichia coli and purified to apparent homogeneity. The purified hTR beta 1 was used to produce monoclonal antibodies. Three hybridomas, secreting mAb J51, J52, and J53, were isolated. All of these mAbs recognized hTR beta 1. J51 and J52 belong to the immunoglobulin G1-k subclass; J53 is an IgM. To evaluate cross-reactivity with other classes of c-erbAs, the three mAbs were used to immunoprecipitate the in vitro translation products of human (h) TR alpha 1, TR alpha 2, rat (r) TR beta 1, TR alpha 1, and TR alpha 2. None of these three mAbs reacted with h- or rTR alpha 1 and TR alpha 2. J51 did not react with rTR beta 1, but J52 and J53 cross-reacted with rTR beta 1 with the same activity as hTR beta 1. To localize the epitopes in the hTR beta 1 molecule, [35S]methionine-labeled and truncated hTR beta 1 containing the hormone-binding domain E (Lys235-Asp456; Lys201-Pro414), domain D (Met169-Asp456), or the DNA-binding domain C (Glu100-Asp456) were expressed in E. coli and purified. Immunoprecipitation of the above truncated hTR beta 1 with mAbs indicated that the epitopes for J51 and J52 were located in two different sites in the A/B domain. The epitope for J53 was located in the E domain. Using immunocytochemistry and mAb J52, the endogenous TR beta 1 in rat pituitary GH3 cells was visualized to be exclusively present in nuclei. The transfected hTR beta 1 in monkey COS-1 and human choriocarcinoma JEG-3 cells was recognized by both J51 and J52. Interestingly, the intracellular localization of the transfected hTR beta 1 or rTR beta 1 in the above two cell lines depended on the level of expression. TR beta 1 expressed at low levels was found exclusively in nuclei. However, for high level expression of TR beta 1, cytoplasmic localization was also detected. J53, however, failed to detect nuclear fluorescence of the endogenous and transfected TR beta 1 in fixed cells, suggesting that its antigenic site might be occluded. Localization of the endogenous and transfected TR beta 1 in nuclei indicated that these two receptor proteins are structurally indistinguishable.(ABSTRACT TRUNCATED AT 400 WORDS)