RESUMO
Cellular differentiation and development of germ cells critically depend on a coordinated activation and repression of specific genes. The underlying regulation mechanisms, however, still lack a lot of understanding. Here, we describe that both the testis-specific transcriptional activator CREMtau (cAMP response element modulator tau) and the repressor GCNF (germ cell nuclear factor) have an overlapping binding site which alone is sufficient to direct cell type-specific expression in vivo in a heterologous promoter context. Expression of the transgene driven by the CREM/GCNF site is detectable in spermatids, but not in any somatic tissue or at any other stages during germ cell differentiation. CREMtau acts as an activator of gene transcription whereas GCNF suppresses this activity. Both factors compete for binding to the same DNA response element. Effective binding of CREM and GCNF highly depends on composition and epigenetic modification of the binding site. We also discovered that CREM and GCNF bind to each other via their DNA binding domains, indicating a complex interaction between the two factors. There are several testis-specific target genes that are regulated by CREM and GCNF in a reciprocal manner, showing a similar activation pattern as during spermatogenesis. Our data indicate that a single common binding site for CREM and GCNF is sufficient to specifically direct gene transcription in a tissue-, cell type- and differentiation-specific manner.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares/metabolismo , Testículo/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cromatina/química , DNA/química , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Elementos de Resposta , Espermátides/metabolismoRESUMO
The acute-phase response (APR) is characterized by an impaired metabolism of the essential trace element selenium (Se). Moreover, low-Se concentrations correlate to mortality risk in sepsis. Therefore, we analyzed the expression of the central Se transport and storage protein selenoprotein P (Sepp1) during an APR in mice. Serum Se and Sepp1 concentrations declined in parallel after injection of lipopolysaccharide to 50 and 39% of control-injected littermates, respectively. This negative APR proceeded largely independent from hepatic Sepp1 transcript concentrations. Instead, we identified a set of hepatic transcripts involved in Se metabolism, which declined coordinately during the APR, including the selenocysteine-specific elongation factor (EFsec), selenophosphate-synthetase 2 (Sephs2), selenocysteine-tRNA[Ser]Sec synthase (SecS), and phosphoseryl-tRNA[Ser]Sec kinase (Pstk). Pstk reacted most strongly and qualified as a new limiting factor for Sepp1 biosynthesis in siRNA-mediated knockdown experiments in hepatocytes in culture. Analogous experiments were performed with mice transgenic for hepatocyte-specific human Sepp1 cDNA to verify this hypothesis. Similar kinetics and effect sizes of Sepp1 expression were observed as before in wild-type mice. We conclude that hepatic translation of Sepp1 mRNA is specifically impaired during the APR. This deficit disrupts regular Se metabolism, transport, and supply to peripheral tissues and likely aggravates the pathological status.
Assuntos
Reação de Fase Aguda/metabolismo , Fígado/metabolismo , Selênio/metabolismo , Selenoproteína P/biossíntese , Animais , Regulação para Baixo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Selenoproteína P/sangue , Selenoproteína P/genéticaRESUMO
BACKGROUND: There is growing evidence that, in addition to the reproductive system, the hypothalamic-pituitary-thyroid axis is a target of endocrine-disrupting compounds (EDCs). However, this is not reflected adequately in current screening and assessment procedures for endocrine activity that to date determine only general parameters of thyroid function. OBJECTIVE AND METHODS: We used several in vitro and ex vivo assays in an attempt to identify suitable biomarkers for antithyroid action testing a selected panel of putative EDCs. RESULTS: In vitro we detected stimulation or inhibition of iodide uptake into FRTL-5 rat thyroid cells, inhibition of thyroid hormone binding to transthyretin, agonistic or antagonistic effects in a thyroid hormone receptor-dependent reporter assay, and inhibition of thyroid peroxidase using a novel assay system based on human recombinant thyroperoxidase that might be suitable for routine screening for potential EDCs. In rats, chronic application of several EDCs led to changes in thyroid morphology, alterations of thyrotropin and thyroid hormone serum levels as well as alterations in peripheral thyroid hormone-regulated end points such as malic enzyme and type I 5'-deiodinase activity. CONCLUSIONS: As the effects of EDCs do not reflect classic mechanisms of hormone-dependent regulation and feedback, we believe multitarget and multimodal actions of EDCs affect the hypothalamic-pituitary-thyroid axis. These complex effects require a diverse approach for screening, evaluation, and risk assessment of potential antithyroid compounds. This approach involves novel in vitro or cell-based screening assays in order to assess thyroid hormone synthesis, transport, metabolism, and action as well as in vivo assays to measure thyroid hormone-regulated tissue-specific and developmental end points in animals.
Assuntos
Disruptores Endócrinos/toxicidade , Glândula Tireoide/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Animais , Biomarcadores , Linhagem Celular , Feminino , Técnicas In Vitro , Iodeto Peroxidase/efeitos dos fármacos , Iodeto Peroxidase/metabolismo , Iodo/metabolismo , Malato Desidrogenase/efeitos dos fármacos , Malato Desidrogenase/metabolismo , Ratos , Ratos Sprague-Dawley , Glândula Tireoide/metabolismo , Tireotropina/sangue , Tireotropina/efeitos dos fármacosRESUMO
To assess interference with endocrine regulation of the thyroid axis, rats (female, ovariectomised) were treated for 12 weeks with the suspected endocrine active compounds (EAC) or endocrine disrupters (ED) 4-nonylphenol (NP), octyl-methoxycinnamate (OMC) and 4-methylbenzylidene-camphor (4-MBC) as well as 17beta-estradiol (E2) and 5alpha-androstane-3beta,17beta-diol (Adiol) on the background of a soy-free or soy-containing diet, and endpoints relevant for regulation via the thyroid axis were measured. Thyrotropin (TSH) and thyroid hormone (T4, T3) serum levels were altered, but not in a way consistent with known mechanisms of feedback regulation of the thyroid axis. In the liver, malic enzyme (ME) activity was significantly increased by E2 and Adiol, slightly by OMC and MBC and decreased by soy, whereas type I 5'-deiodinase (5'DI) was decreased by all treatments. This may be due rather to the estrogenic effect of the ED, as there is no obvious correlation with T4 or T3 serum levels. None of the substances inhibited thyroid peroxidase (TPO) in vitro, except for NP. In general, several endocrine active compounds disrupt the endocrine feedback regulation of the thyroid axis. However, there was no uniform, obvious pattern in the effects of those ED tested, but each compound elicited its own spectrum of alterations, arguing for multiple targets of interference with the complex network of thyroid hormone action and metabolism.
Assuntos
Glândulas Endócrinas/efeitos dos fármacos , Coração/efeitos dos fármacos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Hormônios Tireóideos/sangue , Tireotropina/sangue , Xenobióticos/toxicidade , Animais , Feminino , Iodeto Peroxidase/metabolismo , Malato Desidrogenase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
CONTEXT: Selenium supplementation has been suggested for Hashimoto thyroiditis and Graves' ophthalmopathy. Objective, Design: Our aim is to measure selenium status (p-Se, p-SePP), urine iodine (UI) levels and urine iodine/creatinine ratio (UI/C) in different thyroid diseases (n = 416) from four European countries and to compare the results between patients with and without thyroid autoimmunity. RESULTS: p-Se and p-SePP showed positive correlation and did not correlate with UI/C. Also, these measurements were higher in patients from Italy in comparison with the other countries. Austria had the lowest UI/C ratios. Selenium deficiency exists in these four European countries. Selenium status was lower in patients with Hashimoto thyroiditis and Graves' disease in comparison with non-autoimmune thyroid disease patients and did not differ between autoimmune patients with or without thyroid peroxidase antibodies. The latter correlated positively with age. CONCLUSIONS: Our findings suggest that Se supplementation might have a beneficial effect in autoimmune thyroid patients.
RESUMO
BACKGROUND: Sex-specific differences in blood pressure (BP) suggest an important modulating role of testosterone in the kidney. However, little is known about the interaction between androgens and the mineralocorticoid aldosterone. Our objective was to determine the effects of testosterone in gonadectomized male and female rats on a low-salt diet, and to determine the effect of androgen receptor (AR) blockade by flutamide on BP and on aldosterone levels. METHODS: Normotensive male and female Wistar rats were gonadectomized and put on a low-salt diet. They were treated for 16 days with testosterone or placebo. In addition, the animals received the AR antagonist flutamide or placebo, respectively. BP was measured by tail-cuff method, 24-h urine samples were collected in metabolic cages and blood was collected for hormonal measurements. RESULTS: Testosterone increased BP in males and females, and this effect could be blocked by flutamide. Flutamide treatment itself significantly increased aldosterone levels in male but not in female rats. These elevated aldosterone levels could be lowered by testosterone treatment during AR blockade. Accordingly to aldosterone levels, flutamide increased in males the serum sodium/potassium to urinary sodium/potassium ratio, an in vivo indicator of renal aldosterone action. CONCLUSIONS: Testosterone regulates BP in male and female gonadectomized rats via the AR. Flutamide by itself exerts influence over aldosterone in the absence of gonadal steroid replacement suggesting AR involvement in renal sodium handling. These flutamide effects were sex-specific and not seen in female rats.