Peter Josef Hofmann

Down-regulation of the hepatic selenoprotein biosynthesis machinery impairs selenium metabolism during the acute phase response in mice

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 (Seppl) during an APR in mice. Serum Se and Sepp1 concentrations declined in parallel after injection of lipopolysaccha‐ride 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), andphosphoseryl‐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 forhepato‐cyte‐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.—Renko, K., Hofmann, P.J., Stoedter, M., Hollenbach, B., Behrends,T., Kohrle, J., Schweizer, U., Schomburg, L. Down‐regulation of the hepatic selenoprotein biosynthesis machinery impairs selenium metabolism during the acute phase response in mice. FASEB J. 23, 1758–1765 (2009)

Endocrine disruptors and the thyroid gland--a combined in vitro and in vivo analysis of potential new biomarkers.

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.

Interference of Endocrine Disrupters with Thyroid Hormone Receptor–Dependent Transactivation

Thyroid hormones regulate critical developmental processes and key metabolic pathways. A number of natural and synthetic substances have been identified which adversely interfere with the endocrine system. These so-called endocrine disrupters (ED) have mainly been studied for their impact on the gonadal hormone axis. The aim of this work was to develop a novel sensitive and convenient in vitro screening assay for the detection and characterization of potential ED of thyroid hormone (TH)-dependent transactivation of gene transcription and to apply this tool to test relevant environmental and nutritive ED compounds. We constructed a TH-responsive luciferase-based reporter plasmid and established a reporter gene assay in a 96 well microplate format using the human hepatocarcinoma cell line HepG2 as host system. Both the synthetic TH receptor (TR) agonist GC-1 and the antagonist NH-3 were used to evaluate the assay. Concentration-response data of test compounds (food constituents, isoflavones, ultraviolet-absorbers, pesticides, industrial chemicals) were recorded in activation assays. In addition, interference with TH-mediated transactivation was tested by coincubation of the ED with triiodothyronine (T(3)) in competition assays. Most ED tested affected T(3) reporter gene activity at concentrations of 1 microM or higher and displayed either agonistic or mixed agonistic/antagonistic activities. Effects of relevant ED occurred only at relatively high concentrations compared with the endogenous TR ligand T(3). However, on basis of their high production volumes and potential bioaccumulation of some fat-soluble ED our data indicate the need to carefully monitor certain ED for potential disruption of the TH system in intact organisms and humans.

Functional cooperation between CREM and GCNF directs gene expression in haploid male germ cells

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 CREMτ (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. CREMτ 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.

Identification of thyroid hormone response elements in vivo using mice expressing a tagged thyroid hormone receptor α1

TRα1 (thyroid hormone receptor α1) is well recognized for its importance in brain development. However, due to the difficulties in predicting TREs (thyroid hormone response elements) in silico and the lack of suitable antibodies against TRα1 for ChIP (chromatin immunoprecipitation), only a few direct TRα1 target genes have been identified in the brain. Here we demonstrate that mice expressing a TRα1–GFP (green fluorescent protein) fusion protein from the endogenous TRα locus provide a valuable animal model to identify TRα1 target genes. To this end, we analysed DNA–TRα1 interactions in vivo using ChIP with an anti-GFP antibody. We validated our system using established TREs from neurogranin and hairless, and by verifying additional TREs from known TRα1 target genes in brain and heart. Moreover, our model system enabled the identification of novel TRα1 target genes such as RNF166 (ring finger protein 166). Our results demonstrate that transgenic mice expressing a tagged nuclear receptor constitute a feasible approach to study receptor–DNA interactions in vivo, circumventing the need for specific antibodies. Models like the TRα1–GFP mice may thus pave the way for genome-wide mapping of nuclear receptor-binding sites, and advance the identification of novel target genes in vivo.

Sex-specific effects of spironolactone on blood pressure in gonadectomized male and female Wistar rats.

A low-salt diet is known to decrease and salt excess to increase blood pressure in humans and rodents. Sex steroids seem to play a role in salt dependent hypertension. However, little is known about sex differences in mineralocorticoid receptor blockade between male and female rats. The objective of the work was at first to investigate the effects of a low-salt vs. a high-salt diet on blood pressure without the influence of gonadal steroids in male and female rats. Second, to determine the sex-specific effects of mineralocorticoid receptor blockade by spironolactone in high-salt and low-salt fed gonadectomized male and female animals. Normotensive male and female Wistar rats were gonadectomized and put on a low (NaCl<0.03%) or high (NaCl=4%) salt diet. On each diet animals received spironolactone or placebo. Blood pressure was measured by tail-cuff-method; 24-h urine samples were collected in metabolic cages and blood was collected for hormonal measurements. High-salt diet significantly increased systolic blood pressure in both sexes. This effect could be blocked effectively by spironolactone only in male rats. Spironolactone treatment significantly increased aldosterone levels in males and females independent of the sodium content of the diet. High sodium diet significantly increased relative kidney weight, which was not altered by spironolactone treatment. Independently of gonadal steroids a high-salt diet increased blood pressure in gonadectomized male and female rats. Spironolactone lowered blood pressure only in male not in female rats on a high-salt diet clearly indicating sex-specific effects of the mineralo-corticoid antagonist spironolactone.

Structure- and cell-specific effects of imidoselenocarbamates on selenoprotein expression and activity in liver cells in culture

The essential micronutrient selenium (Se) exerts its biological effects mainly through selenoproteins thereby affecting a number of physiological pathways including intracellular redox control, stress response and cancer cell proliferation. Besides affecting selenoprotein expression, some selenocompounds have been synthesized and analyzed in order to serve as chemotherapeutic substances preferentially targeting cancer cells. This promising chemotherapeutic potential has recently been verified for a particular imidoselenocarbamate in a mouse tumor model. In the present study we tested the effects of this and a number of related Se-methyl- and Se-benzyl-imidoselenocarbamates on selenoprotein expression in nontransformed and hepatic carcinoma cells in culture. Most of the Se-benzyl-imidoselenocarbamates strongly stimulated selenoprotein P (SePP) secretion while the Se-methyl-imidoselenocarbamates elicited less pronounced effects in hepatocarcinoma HepG2 cells. However, most of the Se-methyl-imidoselenocarbamates increased glutathione peroxidase (GPx) activity and decreased thioredoxin reductase (TXNRD) activity in parallel, while the majority of the Se-benzyl-imidoselenocarbamates were without a respective effect in HepG2 cells. Performing inhibitor assays in vitro, GPx activity was unaffected by the imidoselenocarbamates. In contrast, most of the Se-methyl-imidoselenocarbamates inhibited TXNRD activity in vitro in line with the results in HepG2 cells. Both classes of imidoselenocarbamates strongly induced selenoprotein S (SELS) expression without a respective increase in ER stress or unfolded protein response which are known inducers of SELS biosynthesis. Notably, many of these effects were cancer cell-specific, and not observed in nontransformed AML12 hepatocytes. Our results indicate that these novel selenocompounds affect expression and activity of crucial selenoenzymes in a compound- and cell-specific way in hepatocytes. Especially the Se-methyl-imidoselenocarbamates elicit a unique spectrum of activities by stimulating GPx activity, SELS expression and SePP secretion while inhibiting TXNRD activity in hepatocarcinoma cells. These effects represent a promising finding with respect to the identification of therapeutic selenocompounds, as cancer-cell specificity is combined with desired effects on selenoprotein expression and activity.

Flutamide increases aldosterone levels in gonadectomized male but not female Wistar rats.

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.

Selenium status in patients with autoimmune and non-autoimmune thyroid diseases from four European

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.