Enfermedad de Graves con predominio de T3 en la edad pediátrica / T3-predominant Graves' disease in paediatric patients

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Mediator modulates Gli3-dependent Sonic hedgehog signaling.

The physiological and pathological manifestations of Sonic hedgehog (Shh) signaling arise from the specification of unique transcriptional programs dependent upon key nuclear effectors of the Ci/Gli family of transcription factors. However, the underlying mechanism by which Gli proteins regulate target gene transcription in the nucleus remains poorly understood. Here, we identify and characterize a physical and functional interaction between Gli3 and the MED12 subunit within the RNA polymerase II transcriptional Mediator. We show that Gli3 binds to MED12 and intact Mediator both in vitro and in vivo through a Gli3 transactivation domain (MBD; MED12/Mediator-binding domain) whose activity derives from concerted functional interactions with both Mediator and the histone acetyltransferase CBP. Analysis of MBD truncation mutants revealed an excellent correlation between the in vivo activation strength of an MBD derivative and its ability to bind MED12 and intact Mediator in vitro, indicative of a critical functional interaction between the Gli3 MBD and the MED12 interface in Mediator. Disruption of the Gli3-MED12 interaction through dominant-negative interference inhibited, while RNA interference-mediated MED12 depletion enhanced, both MBD transactivation function and Gli3 target gene induction in response to Shh signaling. We propose that activated Gli3 physically targets the MED12 interface within Mediator in order to functionally reverse Mediator-dependent suppression of Shh target gene transcription. These findings thus link MED12 to the modulation of Gli3-dependent Shh signaling and further implicate Mediator in a broad range of developmental and pathological processes driven by Shh signal transduction.

Putative L-triiodothyronine receptors in the liver nuclei of mature tropical toad, Bufo melanostictus.

Thyroid hormones exert a major role in growth and differentiation of almost all types of tissues in animals, particularly in amphibian metamorphosis, through its specific nuclear receptor activation followed by gene expression. However, its function in mature tropical amphibians is less studied. The present study revealed the existence of a single class of specific nuclear receptor(s) in the liver nuclei of mature tropical toad, Bufo melanostictus, with a dissociation constant of (3.7 +/- 0.9) x 10(-10) molar and maximum binding capacity of 0.074 +/- 0.013 pmol/mg DNA. The percentage of relative binding affinities for the specific nuclear L-T3 binding site in the liver nuclei of toad were L-triiodothyronine (L-T3) > triiodothyroacetic acid (TRIAC) > L-thyroxine (L-T4) = tetraiodothyroacetic acid (TETRAC) > 3,3',5'-triiodothyronine (r-T3) > Diiodothyrtonine (L-T2) (100 > 75 > 19.4 = 19.4 > 3.7 > 0.39) and the relative ED50 values (in nanomolar) were 0.33 < 0.44 < 1.7 = 1.7 < 9 < 83.

Glucocorticoid and thyroid hormone receptors in mitochondria of animal cells.

This article concerns the localization of glucocorticoid and thyroid hormone receptors in mitochondria of animal cells. The receptors are discussed in terms of their potential role in the regulation of mitochondrial transcription and energy production by the oxidative phosphorylation pathway, realized both by nuclear-encoded and mitochondrially encoded enzymes. A brief survey of the role of glucocorticoid and thyroid hormones on energy metabolism is presented, followed by a description of the molecular mode of action of these hormones and of the central role of the receptors in regulation of transcription. Subsequently, the structure and characteristics of glucocorticoid and thyroid hormone receptors are described, followed by a section on the effects of glucocorticoid and thyroid hormones on the transcription of mitochondrial and nuclear genes encoding subunits of OXPHOS and by an introduction to the mitochondrial genome and its transcription. A comprehensive description of the data demonstrates the localization of glucocorticoid and thyroid hormone receptors in mitochondria as well as the detection of potential hormone response elements that bind to these receptors. This leads to the conclusion that the receptors potentially play a role in the regulation of transcription of mitochondrial genes. The in organello mitochondrial system, which is capable of sustaining transcription in the absence of nuclear participation, is presented, responding to T3 with increased transcription rates, and the central role of a thyroid receptor isoform in the transcription effect is emphasized. Lastly, possible ways of coordinating nuclear and mitochondrial gene transcription in response to glucocorticoid and thyroid hormones are discussed, the hormones acting directly on the genes of the two compartments by way of common hormone response elements and indirectly on mitochondrial genes by stimulation of nuclear-encoded transcription factors.

Developmental expression analysis of thyroid hormone receptor isoforms reveals new insights into their essential functions in cardiac and skeletal muscles.

Nuclear thyroid hormone (TH) receptors (TR) play a critical role in mediating the diverse actions of TH in development, differentiation, and metabolism of most tissues, but the role of TR isoforms in muscle development and function is unclear. Therefore, we have undertaken a comprehensive expression analysis of TRalpha 1, TRbeta 1, TRbeta 2 (TH binding), and TRalpha 2 (non-TH binding) in functionally distinct porcine muscles during prenatal and postnatal development. Use of a novel and highly sensitive RNase protection assay revealed striking muscle-specific developmental profiles of all four TR isoform mRNAs in cardiac, longissimus, soleus, rhomboideus, and diaphragm. Distribution of TR isoforms varied markedly between muscles; TRalpha expression was considerably greater than TRbeta and there were significant differences in the ratios TRalpha 1:TRalpha 2, and TRbeta 1:TRbeta 2. Together with immunohistochemistry of myosin heavy chain isoforms and data on myogenesis and maturation of the TH axis, these findings provide new evidence that highlights central roles for 1) TRalpha isoforms in fetal myogenesis, 2) the ratio TRalpha 1:TRalpha 2 in determining cardiac and skeletal muscle phenotype and function; 3) TRbeta in maintaining a basal level of cellular response to TH throughout development and a specific maturational function around birth. These findings suggest that events disrupting normal developmental profiles of TR isoforms may impair optimal function of cardiac and skeletal muscles.

Protein profiles and identification of high performance liquid chromatography isolated proteins of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures.

Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53.

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcriptional factors that regulate growth, differentiation, and development. The molecular mechanisms by which TRs mediate these effects are unclear. One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects. In this study, we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53. p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and/or direct protein-protein interaction. We found that the human TR subtype beta 1 (h-TR beta 1) physically interacted with p53 via its DNA binding domain. As a result of this physical interaction, binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner. In transfected cells, wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1. In contrast, mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements. These results indicate the gene regulating activity of TRs was modulated by p53, suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells.

Multiple functions of the TR2-11 orphan receptor in modulating activation of two key cis-acting elements involved in the retinoic acid signal transduction system.

The testicular receptor 2 (TR2) orphan receptor binds to hormone response elements (HREs) consisting of two AGGTCA half-site direct repeat consensus sequences (DR) with various spacing in the following order: DR1 > DR2 > DR5 DR4 DR6 > DR3. When binding to natural HREs, TR2 orphan receptor remains flexible with higher binding affinities to (a) cellular retinol-binding protein II promoter region (CRBPIIp) (DR1), SV40 +55 region (DR2), and retinoic acid response element beta (RARE beta) (DR5) than to (b) NGFI-B response element (NBRE) and also to (c) the palindromic thyroid hormone response element (TREpal). This wide spectrum of HRE recognition sequences suggests possible versatility of the TR2 orphan receptor in cross-talking with other signal transduction systems. Chloramphenicol acetyltransferase (CAT) assay demonstrates that the TR2 orphan receptor competes with CRBPIIp- and RARE beta-CAT gene expression activated by retinoid X receptor alpha (RXR alpha) and retinoic acid receptor alpha (RAR alpha)/RXR alpha heterodimers, respectively. In addition, this suppression may not be mediated by the formation of heterodimers between TR2 orphan receptor and either RXR alpha or RAR alpha. Instead, a minimum of 100-fold higher affinity of the TR2 orphan receptor for CRBPIIp than RXR alpha may explain why the TR2 orphan receptor dominates RXR alpha in CRBPIIp-CAT activation. Together, our data suggest that the TR2 orphan receptor may be a master regulator in modulating the activation of two key HREs, RARE beta and CRBPIIp, involved in the retinoic acid signal transduction pathway.

[The membrane receptor binding of thyroid hormones in rat liver and brain cells in ontogeny]. / Membrannaia retseptsiia tireoidnykh gormonov v kletkakh pecheni i mozga krys v ontogeneze.

We studied changes of phospholipid and fatty acid composition of highly purified plasma membranes of liver and brain cells of rats over time during ontogenesis. Membranes were isolated from the organs of 2-day-old embryos, 6-day-old newborn rats, and adult rats. We examined the binding of thyroxine, a thyroid hormone, by preparations of plasma membranes from liver and brain at these times and have determined any tissue specificity in the development of membrane-mediated thyroxine reception during ontogenesis.

A 43-kDa protein related to c-Erb A alpha 1 is located in the mitochondrial matrix of rat liver.

In order to characterize Sterling's triiodothyronine (T3) mitochondrial receptor using photoaffinity labeling, we observed two specific T3-binding proteins in the inner membrane (28 kDa) and in the matrix (43 kDa) of rat liver mitochondria. Western blots and immunoprecipitation using antibodies raised against the T3-binding domain of the T3 nuclear receptor c-Erb A alpha 1 indicated that at least the 43-kDa protein was c-Erb A alpha 1-related. In addition, gel mobility shift assays demonstrated the occurrence of a c-Erb A alpha 1-related mitochondrial protein that specifically binds to a natural or a palindromic thyroid-responsive element. Moreover, this protein specifically binds to a direct repeat 2 sequence located in the D-loop of the mitochondrial genome. Furthermore, electron microscopy studies allowed the direct observation of a c-Erb A-related protein in mitochondria. Lastly, the relative amounts of the 43-kDa protein related to c-Erb A alpha 1 were in good correlation with the known mitochondrial mass in three typical tissues. Interestingly, expression of a truncated form of the c-Erb A alpha 1 nuclear receptor in CV1 cells was associated with a mitochondrial localization and a stimulation of mitochondrial activity. These results supply evidence of the localization of a member of the nuclear receptor superfamily in the mitochondrial matrix involved in the regulation of mitochondrial activity that could act as a mitochondrial T3-dependent transcription factor.

Expression of the rat alpha 1 thyroid hormone receptor ligand binding domain in Escherichia coli and the use of a ligand-induced conformation change as a method for its purification to homogeneity.

The rat alpha 1 thyroid hormone receptor (rTR alpha 1) mediates hormone-dependent gene regulation by utilizing several distinct structural domains, including those containing DNA and ligand binding sites. Binding of the hormone to the ligand binding domain (TR-LBD) induces conformational changes in the receptor that are involved in affecting the receptor's transcriptional regulatory and other functions. A 33-kDa protein fragment (Met122-Val410) of rTR alpha 1, which includes the entire TR-LBD, was expressed in Escherichia coli, yielding typically 1.5 mg of soluble TR-LBD/liter of bacteria. The protein was purified to > 99% homogeneity with a final yield of 24% by hydrophobic interaction, DEAE anionic exchange, and heparin cationic exchange chromatographic steps. The Kd of the purified TR-LBD for 3,3',5-triiodo-L-thyronine (T3) was 0.06 nM, identical to that for full-length rTR alpha 1. T3 analogs had affinities consistent with values obtained for full-length rTR alpha 1. In all three chromatography steps, TR-LBD prebound to [125I]T3 eluted earlier than the unliganded TR-LBD, like the full-length receptor. These studies indicate that the binding affinity and specificity of the TR-LBD are similar to those of the intact rTR alpha 1 and that the ligand-induced conformational changes occur in the LBD itself. These studies also provide methodology for obtaining milligram quantities of protein useful for biochemical and biophysical studies of the thyroid hormone receptor and its ligand-induced changes.

Genetic dissection of thyroid hormone receptor beta: identification of mutations that separate hormone binding and transcriptional activation.

The thyroid hormone receptors (TR) are members of the nuclear receptor family of ligand-mediated transcription factors. The large region of TR that lies C-terminal to its DNA-binding domain subserves functions of ligand binding, dimerization, and transactivation. Little is known regarding the structural or functional determinants of these processes. We have utilized genetic screening in the yeast Saccharomyces cerevisiae to identify residues involved in these functions. Random mutations of the rat TR beta 1 isoform between amino acid residues 179 and 456 were screened, and mutants with reduced hormone-dependent activation of reporter gene activity were isolated. In this paper we describe the characterization of a class of mutants that exhibit a dissociation between hormone binding and transcriptional activation. These mutants retained hormone binding (> 15% of the wild-type level) yet failed to transactivate a reporter gene. A number of these mutations occurred within the D region, which links the DNA-binding and ligand-binding domains of the receptor. One subset of these mutations abrogated DNA binding, supporting a role of the D region in this process. The remainder retain DNA binding and thus highlight residues critical for receptor activation. In addition, an unexpected group of "superactivator" mutations that led to enhanced hormone-dependent activation in S. cerevisiae were found. These mutations localized to the carboxy-terminal portion of the receptor in a region which contains elements conserved across the superfamily of nuclear receptors. The hormone-dependent phenotype of these superactivator mutations suggests an important role of this segment in ligand-mediated transcriptional activation.

Purification and properties of thyroid hormone receptor beta 1 expressed in Escherichia coli as a fusion protein.

Thyroid hormone receptor binds to specific DNA sequences and acts as a hormone-dependent transcriptional regulator. The protein can form homodimers, or heterodimers with the related 9-cis-retinoic acid receptor (RXR) or retinoic acid receptor (RAR) receptor families, leading to complex patterns of regulation. To obtain relatively large quantities of the receptor for biochemical studies, we have inserted the cDNA for human thyroid receptor beta into a variant of the pGEX vector (pGEX-KG) and produced the protein in Escherichia coli as a fusion with glutathione-S-transferase. Conditions for protein production, isolation on glutathione agarose, and thrombin cleavage to generate active receptor were developed. Final yields were approximately 1 mg/liter of culture. Scatchard plots of 125I-triiodothyronine binding data revealed a single class of sites with a Kd of 0.1 nM. An overlay assay was established to measure protein-protein binding and used to show a direct interaction with bacterially expressed RXR receptor. Binding of the purified receptor to DNA response elements measured in a DNA binding assay was increased by RXR to different extents, depending on the DNA sequence. This preparation will be useful in exploring the mechanisms of receptor activity.

[Isolation and characteristics of the thyroxine membrane receptor]. / Vydelenie i kharakteristika membrannogo retseptora tiroksina.

A thyroxin receptor was isolated from rat liver plasma membranes by means of affinity chromatography. The receptor proved to be glycoprotein with a molecular mass of 40 kDa and contained 322 amino acid residues. Incorporation of the purified receptor into artificial phospholipid membrane was carried out.

Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.

Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors.

Thyroid hormone receptor functions as monomeric ligand-induced transcription factor on octameric half-sites. Consequences also for dimerization.

The thyroid hormone (3,5,3'-triiodothyronine) receptor (T3R) belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. T3Rs are known to bind as homodimers and heterodimers with retinoid X receptors (RXRs) to two hexameric half-sites in directly repeated, palindromic, and inverted palindromic orientations. The binding of T3R monomers to individual half-sites was often reported, but no clear ligand-induced transactivational activity has been shown. Here, we analyzed interactions of T3R monomers with individual half-sites of the sequence NNAGGTCA. We found that the two nucleotides 5' of the AGGTCA core half-site strongly influence T3R binding and transcriptional activity: octameric half-sites of the consensus sequence (T/C)(A/G)AGGTCA were bound by T3Rs with the highest affinity. This suggests T3R functioning also as a monomeric transcription factor like the orphan nuclear receptors NGFI-B and FTZ-F1. Moreover, we observed that the function of T3R-RXR heterodimers on response elements composed of two half-sites in a directly repeated orientation spaced by 4 nucleotides is determined in major parts by the 5'-flanking sequence of the upstream half-site. Consequently, we noted that the affinity of T3R homodimers is influenced by both 5'-flanking sequences. Our findings suggest that the binding of dimerizing receptors like T3R and other nuclear receptors to their cognate response elements is determined not only by the half-site core sequence, orientation, and number of spacing nucleotides, but also by the nucleotide sequence preceding the half-sites.

Preparation of unoccupied thyroid-hormone receptor.

Thyroid hormone (3,5,3'-tri-iodothyronine; T3) regulates gene expression through binding to its specific receptor in the nucleus. In euthyroid animals, roughly half of all receptors are occupied by the hormone. Nuclear extracts thus yield mixtures of occupied and unoccupied receptors. We present here a simple method for transforming occupied receptors into unoccupied ones. In vitro, the T3-receptor complex dissociated in a half-dissociation time exceeding 100 h at 0 degrees C, and at temperatures that accelerated the dissociation the receptor was quickly inactivated. Long-chain-fatty-acyl-CoAs, on the other hand, greatly accelerated the dissociation of T3-receptor complex at 0 degree C. The receptor was extracted from rat liver nuclei, incubated with oleoyl-CoA to release the bound hormone, and passed through a small column of Lipidex, which strongly adsorbed both oleoyl-CoA and the dissociated hormone. The receptor was recovered in the flow-through fraction in its unoccupied form, as seen by the results of DEAE-Sephadex column chromatography and the loss of all previously bound [125I]T3. The maximum T3-binding capacity of the unoccupied receptor was about 1.5-fold that of the untreated sample, and the dissociation constant was unaltered. The results suggest that most nuclear thyroid-hormone receptors occupied by the hormone were transformed into unoccupied ones. From the T3-binding capacity before and after oleoyl-CoA treatment, the in vivo T3 occupancy of the receptor was estimated. The procedure is easy to perform, and the method should be useful for studies of unoccupied receptors.

One-step immunoaffinity purification of human beta 1 thyroid hormone receptor with DNA and hormone binding activity.

An efficient and versatile method to purify large amounts of active human beta 1 thyroid hormone receptor (h-TR beta 1) was developed. Using a T7 expression system, h-TR beta 1 was overexpressed in Escherichia coli. Approx. 80% of the expressed receptor protein was concentrated in the insoluble inclusion bodies and approximately 20% was in the soluble form (h-TR beta 1-S). h-TR beta 1-S was conveniently purified by one immunoaffinity chromatographic step. From 1 l of cell culture, approx. 0.1 mg of purified h-TR beta 1-S was obtained. The purified h-TR beta 1-S binds to 3,3',5-triiodo-L-thyronine with a Ka = 2 x 10(9) M-1 and exhibits analog specificity. The purified h-TR beta 1-S also binds to T3 response elements (TRE) with different orientation in the half-sites with differential activity. In addition, binding of h-TR beta 1-S to TREs was enhanced by retinoid X receptor. These results indicate that the purified h-TR beta 1-S retains its hormone and DNA binding activity. The purified h-TR beta 1-S is suitable for structural and functional studies. This method could be used to purify h-TR beta 1 or rat TR beta 1 expressed in insect cells or yeast.

Arsenical-based affinity chromatography of vicinal dithiol-containing proteins: purification of L1210 leukemia cytoplasmic proteins and the recombinant rat c-erb A beta 1 T3 receptor.

Proteins containing vicinal dithiols were purified by affinity chromatography using Sepharose 4B linked to aminohexanoyl-4-aminophenylarsineoxide (As-Sepharose). The protein vicinal dithiols form stable dithioarsine derivatives with the arsine oxide moieties of the gel. The adsorbed proteins were eluted, at physiological pH, by buffers containing beta-mercaptoethanol or dithiothreitol. The dithiol proteins were identified by their specific labeling with N-iodoacetyl-3-[125I]-iodotyrosine. Cytoplasmic thiol proteins of L1210 murine leukemia lymphoblasts were separated into three classes by interaction with As-Sepharose. Proteins that did not bind to the gel consisted of monothiol proteins; proteins eluted by beta-mercaptoethanol include vicinal dithiol-containing proteins with low affinity for the arsine oxide. DL-Dithiothreitol (DTT) elutes a large group of vicinal dithiol-containing proteins with high affinity for the arsine groups. Gradient elution allowed characterization of the relative affinities of dithiol proteins for the As-Sepharose. A one-step purification of the L-triiodothyronine recombinant rat c-erb A beta 1 T3 receptor synthesized in yeast required pretreatment with DTT for binding to As-Sepharose and resulted in a 62-fold increase in specific activity. The procedure allows purification of proteins inhibited by phenylarsine oxide such as phosphotyrosine phosphatases, proteins that are subject to redox regulation, and dithiol proteins that are targets of oxidative stress.