RESUMO
In newborns and small mammals, cold-induced adaptive (or nonshivering) thermogenesis is produced primarily in brown adipose tissue (BAT). Heat production is stimulated by the sympathetic nervous system, but it has an absolute requirement for thyroid hormone. We used the thyroid hormone receptor-beta--selective (TR-beta--selective) ligand, GC-1, to determine by a pharmacological approach whether adaptive thermogenesis was TR isoform--specific. Hypothyroid mice were treated for 10 days with varying doses of T3 or GC-1. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was restored by either T3 or GC-1 treatment. However, whereas interscapular BAT in T3-treated mice showed a 3.0 degrees C elevation upon infusion of norepinephrine, indicating normal thermogenesis, the temperature did not increase (<0.5 degrees C) in GC-1--treated mice. When exposed to cold (4 degrees C), GC-1--treated mice also failed to maintain core body temperature and had reduced stimulation of BAT UCP1 mRNA, indicating impaired adrenergic responsiveness. Brown adipocytes isolated from hypothyroid mice replaced with T3, but not from those replaced with GC-1, had normal cAMP production in response to adrenergic stimulation in vitro. We conclude that two distinct thyroid-dependent pathways, stimulation of UCP1 and augmentation of adrenergic responsiveness, are mediated by different TR isoforms in the same tissue.
Assuntos
Acetatos/farmacologia , Tecido Adiposo Marrom/fisiologia , Fenóis/farmacologia , Isoformas de Proteínas/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Termogênese/fisiologia , Hormônios Tireóideos/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Temperatura Baixa , AMP Cíclico/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Glicerolfosfato Desidrogenase/biossíntese , Glicerolfosfato Desidrogenase/genética , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipotireoidismo/complicações , Hipotireoidismo/tratamento farmacológico , Hipotireoidismo/fisiopatologia , Canais Iônicos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Malato Desidrogenase/biossíntese , Malato Desidrogenase/genética , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais , Norepinefrina/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Ratos , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Termogênese/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Tri-Iodotironina/uso terapêutico , Proteína Desacopladora 1RESUMO
The sodium/iodide symporter (NIS) mediates iodide uptake in the thyroid gland and lactating breast. NIS mRNA and protein expression are detected in most thyroid cancer specimens, although functional iodide uptake is usually reduced resulting in the characteristic finding of a 'cold' or non-functioning lesion on a radioiodine image. Iodide uptake after thyroid stimulating hormone (TSH) stimulation, however, is sufficient in most differentiated thyroid cancer to utilize beta-emitting radioactive iodide for the treatment of residual and metastatic disease. Elevated serum TSH, achieved by thyroid hormone withdrawal in athyreotic patients or after recombinant human thyrotropin administration, directly stimulates NIS gene expression and/or NIS trafficking to the plasma membrane, increasing radioiodide uptake. Approximately 10-20% differentiated thyroid cancers, however, do not express the NIS gene despite TSH stimulation. These tumors are generally associated with a poor prognosis. Reduced NIS gene expression in thyroid cancer is likely due in part, to impaired trans-activation at the proximal promoter and/or the upstream enhancer. Basal NIS gene expression is detected in about 80% breast cancer specimens, but the fraction with functional iodide transport is relatively low. Lactogenic hormones and various nuclear hormone receptor ligands increase iodide uptake in breast cancer cells in vitro, but TSH has no effect. A wide range of 'differentiation' agents have been utilized to stimulate NIS expression in thyroid and breast cancer using in vitro and in vivo models, and a few have been used in clinical studies. Retinoic acid has been used to stimulate NIS expression in both thyroid and breast cancer. There are similarities and differences in NIS gene regulation and expression in thyroid and breast cancer. The various agents used to enhance NIS expression in thyroid and breast cancer will be reviewed with a focus on the mechanism of action. Agents that promote tumor differentiation, or directly stimulate NIS gene expression, may result in iodine concentration in 'scan-negative' thyroid cancer and some breast cancer.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Simportadores/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Humanos , Iodetos/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , Retinoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Simportadores/efeitos dos fármacos , Tireotropina/fisiologiaRESUMO
While external ionizing radiation has been used for treating non-small cell lung cancer (NSCLC), improved efficacy of this modality would be an important advance. Ectopic expression of the sodium iodide symporter (NIS) and thyroperoxidase (TPO) genes in NSCLC cells facilitated concentration of iodide in NSCLC cells, which markedly induced apoptosis in vitro and in vivo. Pre-incubation of the NIS/TPO-modified NSCLC cells in iodide followed by ionizing radiation generates bystander tumoricidal effects and potently enhances tumor cell killing. This iodide-induced bystander effect is associated with enhanced gap junction intercellular communication (GJIC) activity and increased connexin-43 (Cx43) expression. Thus, iodide may serve as an enhancer to markedly improve the efficacy of radiation therapy in combined therapeutic modalities.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Morte Celular/efeitos da radiação , Iodetos/farmacologia , Neoplasias Pulmonares/metabolismo , Apoptose/efeitos dos fármacos , Autoantígenos/metabolismo , Autoantígenos/farmacologia , Efeito Espectador , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Conexina 43/metabolismo , Terapia Genética , Humanos , Iodeto Peroxidase/metabolismo , Iodeto Peroxidase/farmacologia , Iodetos/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Ligação ao Ferro/farmacologia , Neoplasias Pulmonares/genética , Radiação Ionizante , Sensibilidade e Especificidade , Simportadores/metabolismo , TransfecçãoRESUMO
The thyroid hormone receptor genes, TRalpha and TRbeta, differ in developmental expression and tissue distribution. TRbeta knockout mice have goiter, elevated thyroid hormone and TSH levels, and a functional auditory defect. In contrast, mice with TRalpha 1/alpha2 inactivation have thyroid hypoplasia, low serum thyroid hormone levels, growth arrest and delayed small intestine maturation. Mice with selective TRalpha1 inactivation have apparent normal growth and development, but have bradycardia and reduced body temperature. The dramatic differences between these mice with TRbeta and TRalpha gene inactivations indicate the differential function of these genes. The influence of these gene inactivations on thyroid-stimulating hormone regulation is central to the resulting phenotypes.
RESUMO
We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.
Assuntos
AMP Cíclico/farmacologia , Dexametasona/farmacologia , Hormônio do Crescimento/genética , Regiões Promotoras Genéticas , Tri-Iodotironina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Bovinos , Humanos , Neoplasias Hipofisárias , Ratos , TransfecçãoRESUMO
The heterogeneity of tissue-specific manifestations of generalized resistance to thyroid hormone (GRTH) could result from differential interactions between the mutant thyroid hormone (T3) receptor-beta (TR beta) on T3 response elements (TREs) in different T3-responsive genes. To explore this hypothesis, the mutant TR beta associated with kindred A, P448H; a TR beta mutant, P448L; and a comparable TR alpha mutant (P398H) were tested for intrinsic function and for inhibition of wild-type TR alpha- and -beta-induced expression from four structurally distinct TREs, the rGH ABC*, the rGH palindrome (PAL), the rat malic enzyme (ME), and the chicken lysozyme silencer F2 (F2). The relative function of the mutants was similarly reduced on the four TREs studied and was T3 concentration dependent. The TR alpha mutant retained the intrinsically greater potency characteristic of this isoform, but remained impaired with respect to wild-type TR alpha even at 500 nM T3. In general, dominant negative inhibition of wild-type TR alpha and -beta function was dependent upon the T3 concentration, as expected from the decreased affinity for ligand conferred by this mutation. A T3 concentration sufficient to relieve the inhibition of wild-type TR function on the ABC*, PAL, and ME TREs (50 nM) had no effect on inhibition of the F2 TRE by the mutant TRs. Receptor isoform preferential inhibition was observed on the ABC*, PAL, and ME TREs by the mutant TRs. Thus, both TRE structure and the isoform of endogenously active receptor could determine the degree of inhibition of a specific gene in GRTH individuals. Further, the lack of dominant negative potentials does not explain the absence of TR alpha mutations in GRTH kindreds.
Assuntos
DNA/metabolismo , Mutação , Receptores dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/farmacologia , Animais , Sequência de Bases , Linhagem Celular , Galinhas , Cloranfenicol O-Acetiltransferase/genética , Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/genética , Histidina/genética , Malato Desidrogenase/genética , Camundongos , Dados de Sequência Molecular , Muramidase/genética , Mutagênese Sítio-Dirigida , Prolina/genética , Ratos , Receptores dos Hormônios Tireóideos/genética , Sequências Repetitivas de Ácido Nucleico , TransfecçãoRESUMO
Thyroid hormone (T3) and retinoic acid (RA) are essential for normal vertebrate development and are known to coregulate several genes. Early development is predominantly retinoic acid sensitive, yet thyroid hormone receptor-alpha (T3R alpha) is expressed along with retinoic acid receptors (RAR)-alpha, -beta, and -gamma. To determine the role of unliganded T3R alpha in early development and on RA-stimulated neural development, we used homologous recombination techniques to inactivate both T3R alpha gene alleles in mouse embryonic stem (ES) cells. Loss of both T3R alpha alleles resulted in an increase in basal and RA-induced expression of the endogenous RA-responsive genes, RAR beta and alkaline phosphatase, which demonstrates that T3R alpha has an inhibitory effect on the RA response. A similar magnitude of T3R inhibition of the RA response was seen in transient transfection assays of RA response elements in both ES and assays of RA response elements in both ES and JEG cells. Cotransfection experiments were used to demonstrate that inhibition of the RA response could be mediated by T3R alpha 1. The addition of T3R alpha 1, but not the T3R alpha variant c-erbA alpha 2, to T3R alpha-null ES cells restored the inhibitory effect on RA-induced gene expression. RA-stimulated neural differentiation was seen in the wild-type, but not in T3R alpha-null ES, cells, consistent with reports of abnormal neural development as a consequence of premature RA stimulation. Our results demonstrate that the early expression of unliganded T3R alpha functions to modulate the RA response and RA-stimulated neural differentiation.
Assuntos
Fosfatase Alcalina/genética , Neurônios/citologia , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Células-Tronco/citologia , Tretinoína/farmacologia , Fosfatase Alcalina/análise , Fosfatase Alcalina/metabolismo , Alelos , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , DNA/análise , DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Camundongos , Neurônios/química , Neurônios/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores do Ácido Retinoico/análise , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Homologia de Sequência do Ácido Nucleico , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , TransfecçãoRESUMO
Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.
Assuntos
Proteínas de Transporte/fisiologia , Regiões Promotoras Genéticas , Receptores dos Hormônios Tireóideos/fisiologia , Ativação Transcricional , Tretinoína/farmacologia , Tri-Iodotironina/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/efeitos dos fármacos , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neoplasias Hipofisárias , Plasmídeos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Receptores do Ácido Retinoico , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
We have previously identified sequences required for thyroid hormone (T3) induction of the rat GH (rGH) promoter, which lie in a region from -188 to -164 upstream of the mRNA start site. Within this region, Domains A, -189 to -184 and B, -179 to -174, are imperfect direct repeats, and domain C, -172 to -167, is a divergent inverted copy that matches the A domain at 4/6 positions. A series of synthetic mutant versions of this sequence were inserted upstream of a truncated rGH promoter, or as a replacement for wild-type sequences in a synthetic 237 base pair rGH promoter or upstream of the heterologous thymidine kinase promoter. Mutations changing the B domain to a perfect copy of the A domain significantly increased T3 induction (21.3-fold) relative to the wild type (3.6-fold). A single point mutation making the C domain a better match to the A domain also increased T3 induction to 16.2-fold. Combining this up-mutation with any of three down-mutations in the A, B, or C domains strongly decreased response, showing that all three domains contribute to the amplified T3 response. Binding affinity of the various mutant oligonucleotides was assessed using in vitro translated receptor and affinity paralleled the functional responses for most binding site mutations. Requirements for in vitro binding were, however, less rigorous than those for functional T3 induction. Based on these results, we propose a consensus T3 receptor binding half-site, AGGT(C/A)A, at least two copies of which are required for a T3 response.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hormônio do Crescimento/genética , Mutação , Regiões Promotoras Genéticas/efeitos dos fármacos , Hormônios Tireóideos/genética , Animais , Sequência de Bases , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/farmacologiaRESUMO
Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Receptores dos Hormônios Tireóideos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Galinhas , Clonagem Molecular , Escherichia coli/genética , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos , Plasmídeos , Ratos , Receptores dos Hormônios Tireóideos/genética , Sequências Repetitivas de Ácido Nucleico , TransfecçãoRESUMO
The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat beta TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 basepairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat beta TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hormônio do Crescimento/biossíntese , Regiões Promotoras Genéticas , Tri-Iodotironina/farmacologia , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas/genética , Ratos , Tireotropina/biossíntese , Tireotropina/genética , TransfecçãoRESUMO
The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2-7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.
Assuntos
Carcinoma Papilar/genética , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Regiões Promotoras Genéticas/fisiologia , Simportadores , Glândula Tireoide/fisiologia , Neoplasias da Glândula Tireoide/genética , Sequência de Bases/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Genoma , Humanos , Iodetos/metabolismo , Iodetos/farmacocinética , Mutação , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Fragmentos de Peptídeos/genética , RNA Mensageiro/metabolismo , Valores de Referência , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fator Nuclear 1 de Tireoide , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Thyroid hormone receptor (T3R)-accessory protein heterodimers preferentially bind to thyroid hormone response elements (TREs), which contain hexamer domains arranged as a direct repeat separated by a 4-basepair gap (DR + 4). T3R homodimers, however, preferentially bind to elements that consist of an inverted palindrome. We now report on unique T3R binding patterns and functional characteristics of two such elements that mediate T3 regulation. We performed mutational analysis of the chicken lysozyme silencer F2 (LysF2) TRE and demonstrated that the two functional binding domains are arranged as an inverted palindrome separated by 6 basepairs. Both the LysF2 TRE and the similarly arranged myelin basic protein TRE bind T3R dimers at very low T3R concentrations. Despite the high relative affinity for T3R dimer binding, the T3 induction conferred by these elements is low compared to that of previously characterized TREs with a DR + 4 arrangement. The laminin-B1 gene element, previously shown to bind retinoic acid receptor, contains at least four hexameric binding domains. All of the domains can bind T3R simultaneously and are involved in conferring T3 induction, but bind with a different pattern than that reported for retinoic acid receptor. T3R homodimer binding to a series of mutant laminin-B1 elements and T3 induction were significantly correlated (r = 0.82; P < 0.05). T3R homodimer binding to LysF2 element mutants was not correlated with T3 induction (r = 0.32; P > 0.05); however, T3R-nuclear protein heterodimer binding was significantly correlated (r = 0.67; P < 0.05). T3R-nuclear protein heterodimers, but not homodimers, bound consistently to mutations of the LysF2 element that altered the gap between hexamers. The overall discordance between strong T3R binding to these elements and weak T3 induction indicates that the unusual hexamer arrangement places the T3R complex in an unfavorable configuration for maximal T3-dependent transactivation. The differential T3 sensitivity of generalized resistance to thyroid hormone-associated T3R mutants to the LysF2 element compared with the DR + 4 arrangement suggests that these unique features may have physiological significance.
Assuntos
Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , Galinhas , Dados de Sequência Molecular , Muramidase/genética , Mutação , Receptores dos Hormônios Tireóideos/química , Tri-Iodotironina/farmacologiaRESUMO
The majority of prohormones are cleaved at paired basic residues to generate bioactive hormones by prohormone convertases (PCs). As PC1 and PC2, two neuroendocrine-specific PCs, appear to be the key enzymes capable of processing a variety of prohormones, alterations of PC2 and/or PC1 levels will probably have a profound effect on hormonal homeostasis. We investigated the regulation of PC2 messenger RNA (mRNA) by thyroid hormone using GH3 cells to demonstrate that T3 negatively regulated PC2 mRNA levels in a dose- and time-dependent fashion. Functional analysis of progressive 5'-deletions of the human (h) PC2 promoter luciferase constructs in GH3 cells demonstrated that the regulation probably occurs at the transcriptional level, and that putative negative thyroid hormone response elements were located within the region from -44 to + 137 bp relative to the transcriptional start site. Transient transfections in JEG-3 cells and COS-1 cells showed that the suppressive effect of T3 was equally mediated by the thyroid hormone receptor (TR) isoforms TRalpha1 and TRbeta1. Electrophoretic mobility shift assays using purified TRal and retinoid X receptor-beta protein as well as GH3 nuclear extracts showed that regions from +51 to +71 bp and from +118 to +137 bp of the hPC2 promoter bind to TRalpha1 as both a monomer and a homodimer and with TRalpha1/retinoid X receptor-beta as a heterodimer. Finally, the in vivo regulation of pituitary PC2 mRNA by thyroid status was demonstrated in rats. These results demonstrate that T3 negatively regulates PC2 expression at the transcriptional level and that functional negative thyroid hormone response elements exist in the hPC2 promoter. We postulate that the alterations of PC2 activity may mediate some of the pathophysiological consequences of hypo- or hyperthyroidism.
Assuntos
Regiões Promotoras Genéticas/genética , Receptores dos Hormônios Tireóideos/genética , Subtilisinas/genética , Animais , Antitireóideos/farmacologia , Northern Blotting , Linhagem Celular , Eletroforese , Deleção de Genes , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/genética , Humanos , Isotretinoína/farmacologia , Luciferases/genética , Masculino , Plasmídeos/genética , Pró-Proteína Convertase 2 , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Tri-Iodotironina/farmacologia , Tri-Iodotironina/fisiologiaRESUMO
Retinoid-X receptor (RXR) forms heterodimers with thyroid hormone receptor (TR) and significantly enhances binding to thyroid hormone response elements (TREs). Expression of RXR in a transient transfection assay augments the T3 response, but the influences of the specific cell line and TRE used have not been systematically studied. We determined RXR alpha and -beta augmentation of the TR alpha-mediated T3 response in transient transfection assays of COS, JEG, and mouse embryonic stem (ES) cell lines for a series of eight wild-type thyroid hormone (T3) and retinoic acid response elements (previously shown to bind TR). RXR augmented T3-induced expression in COS and ES cells (1.5- to 4-fold greater expression with added RXR compared to TR alone), but had minimal effect on augmentation of response in JEG cells. For most elements studied there was a proportional augmentation of basal and T3-stimulated expression. TREs from rat GH and laminin-B1, however, had relatively higher levels of T3-induced expression as a result of RXR cotransfection (T3 induction ratios increased 2-fold or greater). Previous characterization of these elements demonstrates that they contain more than two hexameric binding domains, all of which can simultaneously bind TR. The influence of endogenous RXR expression in a cell line on RXR augmentation of the T3 response was determined. RXR alpha and -beta messenger RNA (mRNA) expression was quantitated by Northern blot in each cell line. COS and JEG cells expressed almost exclusively RXR alpha mRNA, although expression was almost 2-fold higher in JEG compared to COS cells (12 +/- 2.5 vs. 6.8 +/- 0.5 density units relative to actin; mean +/- SE; P < 0.05). ES cells expressed only RXR beta mRNA, but at a very low level (0.4 +/- 0.1). Nuclear extracts prepared from JEG and COS cells augmented TR binding proportional to the endogenous RXR mRNA expression, and the heterodimer band was supershifted by the addition of antibody to RXR alpha. Nuclear extracts from ES cells had no detectable TR heterodimer binding to a range of response elements. RXR augmentation of the T3 response differs among cell lines and is greater in those with reduced endogenous RXR. Furthermore, the functional augmentation of the T3 response ratio by RXR is likely to require additional sequences contained in only a subset of elements in which RXR augments TR binding.
Assuntos
Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Ensaio Radioligante , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Receptores X de Retinoides , Alinhamento de Sequência , Células-Tronco , Fatores de Transcrição/genética , TransfecçãoRESUMO
The chicken lysozyme silencer F2 (F2) thyroid hormone response element (TRE) contains an unusual everted palindromic arrangement, has a high affinity for thyroid hormone receptor (TR) homodimers, and is especially sensitive to dominant negative inhibition by, the T3 resistance (RTH) mutant TR beta P453H. We used various TREs and TR mutations to determine the mechanisms for this sensitivity. Changing the F2 orientation from an everted palindrome to a direct repeat with a 4-bp gap (DR+4) (F2-DR) decreased the sensitivity to inhibition at high T3 concentrations, while a loss of this sensitivity occurred with a palindromic arrangement of these same half-sites. F2 contains the dinucleotide TG 5' to each consensus half-site conforming to the optimal TR-binding octamer, YRRGGTCA. A T to A change in position 1 of both F2 half-sites markedly reduced T3-induction, yet only slightly reduced TR homodimer or TR-retinoid X receptor (RXR) heterodimer binding. The TR beta ninth heptad mutation, L428R, prevents TR heterodimerization with RXR and eliminates the inhibitory effect of the P453H mutant TR on the F2-DR, but not the F2 element. Structural features of a TRE that favor strong TR binding of both TR homodimers and TR-RXR heterodimers containing the mutant TR, such as the everted palindromic conformation or the optimal TR-binding consensus octamer, enhance the sensitivity of a TRE to inhibition by the mutant TR. Thus, both half-site orientation and sequence contribute to the sensitivity of a given TRE to dominant negative inhibition by a mutant TR.
Assuntos
Receptores dos Hormônios Tireóideos/antagonistas & inibidores , Receptores dos Hormônios Tireóideos/biossíntese , Sequências Reguladoras de Ácido Nucleico , Tri-Iodotironina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Galinhas , Cloranfenicol O-Acetiltransferase/biossíntese , Sequência Consenso , Resistência a Medicamentos , Rearranjo Gênico , Hormônio do Crescimento/biossíntese , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Sequências Repetitivas de Ácido Nucleico , TransfecçãoRESUMO
A randomized prospective study was done to assess the response of hypothyroxinemic patients with severe nonthyroidal illnesses to T4 therapy. Patients admitted to a medical intensive care unit who had a total serum T4 concentration less than 5 micrograms/dl were randomly assigned to a control (12 patients) or a T4 treatment group (11 patients). L-T4 in a dose of 1.5 micrograms/kg was given iv each day for 2 weeks. In the treatment group, serum T4 and free T4 concentrations significantly increased by day 3 and were normal on day 5. Serum TSH levels decreased significantly in the T4 treatment group, as did the TSH response to TRH. A significant rise in serum T3 occurred in the control group on day 7, but was delayed until day 10 in the treatment group. Mortality was equivalent in the 2 groups (75% control vs. 73% treatment). Regardless of group assignment, survivors and nonsurvivors were completely separable based on baseline T3 to T4 ratios [17.0 +/- 1.8 (+/- SE) ng/micrograms in survivors vs. 7.0 +/- 0.7 in nonsurvivors; P less than 0.001]. Angiotensin-converting enzyme was significantly reduced in the T4 treatment group, but did not rise significantly in response to treatment. T4 therapy was not beneficial in this population of intensive care unit patients, and by inhibiting TSH secretion, it may suppress an important mechanism for normalization of thyroid function during recovery.
Assuntos
Tiroxina/sangue , Tiroxina/uso terapêutico , Adulto , Idoso , Cuidados Críticos , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade , Peptidil Dipeptidase A/sangue , Estudos Prospectivos , Distribuição Aleatória , Talco , Tireotropina/sangue , Hormônio Liberador de Tireotropina , Tri-Iodotironina/sangueRESUMO
Thyroid hormones may alter red blood cell (RBC) sodium content and transport. The functional importance of lithium-sodium (Li-Na) countertransport in regulating sodium (Na) transport in vascular smooth muscle and kidney by Na-H countertransport and the potential effect of thyroid hormone on these processes led us to study Li-Na countertransport and other sodium transporters in RBCs of patients with thyroid dysfunction. Patients with untreated hypothyroidism (10) and hyperthyroidism (10) were studied, along with normal subjects (10). The mean value for Li-Na countertransport was significantly higher in the hypothyroid group [0.46 +/- 0.08 (+/- SE) mmol/L cell.h; P less than 0.05] and lower in the hyperthyroid group (0.15 +/- 0.04 mmol/L cell.h; P less than 0.05) compared to that in the normal subjects (0.25 +/- 0.03 mmol/L cell.h). When all groups were combined, significant negative correlations were found between Li-Na countertransport and serum T4 (r = -0.48; P less than 0.01), free T4 index (r = -0.42; P less than 0.05), and serum T3 (r = -0.38; P less than 0.05). Li-Na countertransport was positively correlated with serum triglyceride (r = 0.57; P less than 0.01), but not with serum cholesterol levels (r = 0.28; P = NS). The values became normal in subsets of the hypothyroid (n = 5) and hyperthyroid groups (n = 5) during treatment. We found a bidirectional effect of thyroid status on RBC Li-Na countertransport, which was reversible when serum thyroid hormone levels became normal. Changes in Li-Na countertransport, a pathway of Na-H exchange, may influence renal sodium handling and vascular tone in patients with thyroid disease and contribute to abnormalities such as hypertension that occur in patients with hypothyroidism.
Assuntos
Eritrócitos/metabolismo , Lítio/farmacocinética , Sódio/metabolismo , Doenças da Glândula Tireoide/sangue , Transporte Biológico/efeitos dos fármacos , Permeabilidade da Membrana Celular , Humanos , Hipertensão/sangue , Hipertensão/etiologia , Hipertireoidismo/sangue , Hipotireoidismo/sangue , Técnicas In Vitro , Potássio/metabolismo , Hormônios Tireóideos/sangue , Hormônios Tireóideos/fisiologia , Triglicerídeos/sangueRESUMO
We employed variable rates of infusion of EDTA or calcium gluconate to examine the relationships between the concentration and rate of change of serum total and plasma ionized calcium and serum immunoreactive intact PTH concentrations in normal subjects. Use of a sensitive immunoradiometric assay specific for PTH-(1-84) made it possible to determine the full range of PTH responses to perturbations in the extracellular calcium concentration. By progressively increasing the rate of administration of calcium gluconate or EDTA during rapid or slow infusions, linear rates of change in the serum calcium concentration were achieved in eight men. The slopes were 2-fold greater during the rapid infusions. Both the calcium infusions decreased serum PTH-(1-84) levels from a mean of 23.2 +/- 2.0 (+/- SE) to 6.4 +/- 1.0 and 5.6 +/- 1.0 ng/L for the rapid and slow infusions, respectively, with no obvious rate dependence. The rapid or slow EDTA infusions increased serum PTH levels to the same maximal extent (95.0 +/- 20.2 and 99.9 +/- 14.5 ng/L, respectively), also with no significant rate dependence. Thus, there was no effect of the rate of change of calcium on the PTH response, which was reflected in similar inverse sigmoidal relationships between PTH and serum total and plasma ionized calcium when the data for the slow and rapid infusions were pooled separately. Similar sigmoidal curves were found in normal women. These data suggest the feasibility of using calcium and EDTA infusions combined with an intact PTH assay to define the relationships between circulating levels of PTH-(1-84) and calcium in states of normal and deranged parathyroid physiology.
Assuntos
Cálcio/sangue , Hormônio Paratireóideo/sangue , Adulto , Gluconato de Cálcio/administração & dosagem , Relação Dose-Resposta a Droga , Ácido Edético/administração & dosagem , Eletrólitos/sangue , Humanos , Infusões Intravenosas , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Vitamina D/sangueRESUMO
Continuous iv administration of ACTH leads to a sustained stimulation of cortisol but a transient stimulation of aldosterone followed by a decline to prestimulation levels by 72 h. Since CRH and ACTH are released in a pulsatile pattern in man, this study sought to investigate whether pulsatile administration of alpha-cosyntropin-(1-24) would lead to the maintenance of aldosterone stimulation over time. Eight normal male subjects on a 10-meq sodium, 100-meq potassium diet received both a continuous and a pulsatile (0.33 U ACTH/pulse over 15 min, pulsed every 2 h) infusion of cosyntropin (4 U/24 h) for 48 h (n = 4) or 72 h (n = 4). Aldosterone and cortisol were sampled every 6 h, and PRA and angiotensin-II every 24 h. Continuous infusion led to a stimulation of aldosterone followed by a progressive decline to preinfusion levels by 72 h [preinfusion 29 +/- 5 ng/dL (810 +/- 139 pmol/L); 72 h, 38 +/- 10 ng/dL (1054 +/- 277 pmol/L); P = 0.40]. Pulsatile infusion led to a stimulation of aldosterone which was maintained up to 72 h [preinfusion 33 +/- 7 ng/dL (915 +/- 194 pmol/L); 72 h, 85 +/- 13 ng/dL (2358 +/- 361 pmol/L); P less than 0.05]. Regression analysis of aldosterone (y) over time (x) from the peak level at 18 h for the continuous infusion showed a significant negative relation (r = 0.63; P = 0.001), indicating a progressive decline in aldosterone. However, for the pulsatile infusion, there was no relation (r = 0.02; P = 0.85), indicating maintenance of aldosterone levels. There were no significant differences in sodium, potassium, PRA, angiotensin-II, or cortisol between infusions to explain these differences in aldosterone levels. Therefore, pulsatile infusion of cosyntropin maintains aldosterone secretion over time.