Gene-specific regulation of hepatic selenoprotein expression by interleukin-6.

Sepsis is a severe inflammatory disease resulting in excessive production of pro-inflammatory cytokines including interleukin-6 (IL-6), causing oxidative stress, tissue damage and organ dysfunction. Health benefits have been observed upon selenium (Se) supplementation in severe sepsis. Selenium is incorporated into selenoproteins implicated in anti-oxidative defence, thyroid hormone metabolism and immunoregulation. Selenium metabolism is controlled by hepatocytes synthesizing and secreting the Se transporter selenoprotein P (SePP). The circulating SePP declines in sepsis causing low serum Se levels. Dysregulation of the hepatic selenoenzyme deiodinase type 1 (DIO1) potentially contributes to the low T3 (thyroid hormone) syndrome observed in severe diseases. We hypothesized that IL-6 affects hepatic selenoprotein biosynthesis directly. Testing human hepatocytes in culture, IL-6 reduced the concentrations of SePP mRNA and secreted SePP in a dose-dependent manner. In parallel, expression of DIO1 declined at the mRNA, protein and enzyme activity level. The effects of IL-6 on glutathione peroxidase (GPX) expression were isozyme-specific; GPX1 remained unaffected, while transcript concentrations of GPX2 increased and those of GPX4 decreased. This pattern of IL-6-dependent effects was mirrored in reporter gene experiments with SePP, DIO1, GPX1, and GPX2 promoter constructs pointing to direct transcriptional effects of IL-6. The redirection of hepatic selenoprotein biosynthesis by IL-6 may represent a central regulatory circuit responsible for the decline of serum Se and low T3 concentrations in sepsis. Accordingly, therapeutic IL-6 targeting may be effective for improving the Se and thyroid hormone status, adjuvant Se supplementation success and survival in sepsis.

Revised reference values for selenium intake.

The German, Austrian and Swiss nutrition societies are the joint editors of the 'reference values for nutrient intake'. They have revised the reference values for the intake of selenium and published them in February 2015. The saturation of selenoprotein P (SePP) in plasma is used as a criterion for the derivation of reference values for selenium intake in adults. For persons from selenium-deficient regions (China) SePP saturation was achieved with a daily intake of 49µg of selenium. When using the reference body weights the D-A-CH reference values are based upon, the resulting estimated value for selenium intake is 70µg/day for men and 60µg/day for women. The estimated value for selenium intake for children and adolescents is extrapolated using the estimated value for adults in relation to body weight. For infants aged 0 to under 4 months the estimated value of 10µg/day was derived from the basis of selenium intake via breast milk. For infants aged 4 to under 12 months this estimated value was used and taking into account the differences regarding body weight an estimated value of 15µg/day was derived. For lactating women compared to non-lactating women a higher reference value of 75µg/day is indicated due to the release of selenium with breast milk. The additional selenium requirement for pregnant women is negligible, so that no increased reference value is indicated.

Strong induction of iodothyronine deiodinases by chemotherapeutic selenocompounds.

The biological activity of thyroid hormones (TH) is regulated by selenoenzymes of the iodothyronine deiodinase (DIO) family catalysing TH activating and inactivating reactions. Besides TH metabolism, several studies indicate an important role of DIO isoenzymes in tumorigenesis and cancer growth. It is therefore of therapeutic importance to identify modulators of DIO expression. We have synthesized and studied a series of selenocompounds containing a methyl- or benzyl-imidoselenocarbamate backbone. One of these novel compounds had chemotherapeutic activities in a murine xenograft tumour model by an unknown mechanism. Therefore, we tested their effects on DIO expression in vitro. In HepG2 hepatocarcinoma cells, DIO1 activity was strongly (up to 10-fold) increased by the methyl- but not by the corresponding benzyl-imidoselenocarbamates. Steady-state mRNA levels remained unaltered under these conditions indicating a post-transcriptional mode of action. The effects were further characterized in HEK293 cells stably expressing DIO1, DIO2 or DIO3. Even within the artificial genetic context of the expression vectors, all three DIO isoenzymes were up-regulated by the methyl- and to a lesser extent by the benzyl-imidoselenocarbamates. Consistent stimulating effects were observed with methyl-N,N'-di(quinolin-3-ylcarbonyl)-imidoselenocarbamate (EI201), a selenocompound known for its anti-tumour activity. DIO inducing effects were unrelated to the intracellular accumulation of selenium, yet the precise mode of action remains elusive. Collectively, our data highlight that these selenocompounds may constitute interesting pharmacological compounds for modifying DIO expression potentially affecting the balance between cell differentiation and proliferation.

[Selenium: benefits and risks]. / Selen: der schmale Grat zwischen Nutzen und Risiko. Nur gezielt supplementieren!

Currently, the results of clinical studies suggest that an increase in the intake of selenium is associated with health benefits. However, the present emphasis should be on diagnosing and treating selenium deficiency resulting from a poor diet or disease. Here we have well-defined risk groups with a demonstrably elevated selenium requirement, and these patients must be provided with appropriate supplementation. On account of the toxic potential of this element, however, uncontrolled self-medication is not to be recommended. Until further studies have confirmed the data indicating a positive effect of selenium supplementation, this highly potent substance must be applied only with great caution and only selectively in specific situations.

Neuronal and ependymal expression of selenoprotein P in the human brain.

Selenoprotein P (SePP) is central to selenium (Se) metabolism in the mammalian organism. Human SePP contains 10 Se atoms that are covalent constituents of the polypeptide chain incorporated as the rare amino acid selenocysteine (Sec). Since hepatocytes secrete SePP into plasma, SePP is commonly regarded as a Se transport protein, although SePP mRNA is expressed in many organs. Gene targeting of SePP in mice leads to neurological dysfunction resulting from Se deficiency and associated reduction of selenoenzyme activities in the brain. However, more recent data revealed that isolated hepatic SePP deficiency does not alter brain Se levels, suggesting a role for SePP locally expressed in the brain. Some of the best characterized and most abundant selenoenzymes, glutathione peroxidases, thioredoxin reductases, and methionine sulfoxide reductase B, play major roles in the cellular defense against reactive oxygen species. Therefore, it was hypothesized that reduced brain Se bioavailability may be involved in the pathogenesis of neurodegenerative disease and normal ageing. We present evidence that human CSF contains SePP and that the human brain expresses SePP mRNA. Moreover, SePP-like immunoreactivity localizes to neurons and ependymal cells and thus appears strategically situated for maintenance and control of Se-dependent anti-oxidative defense systems.

Selenium and selenoproteins in mammals: extraordinary, essential, enigmatic.

Selenium (Se), once known only for its potential toxicity, is now well established as an essential trace element for mammals. Insufficient Se intake predisposes to and manifests in a variety of diseases. Recent studies have proven that it is the synthesis of selenocysteine (Sec)-containing proteins, designated selenoproteins, which represents an essential prerequisite for regular development and a long and healthy life. New transgenic mouse models analysing those selenoproteins with proven enzymatic functions displayed particular phenotypes and highlighted essential Se-dependent processes in development, growth or against specific challenges. While there is a growing molecular understanding of and general agreement on the importance of sufficiently high Se intake and undisturbed selenoprotein biosynthesis, many of the recently identified selenoproteins are still uncharacterised, and the effects and consequences of supra-physiological Se dosages are not biochemically understood. With the recent definition of the human and mouse selenoproteomes and a growing number of available tools, the Se field is now geared for a great leap forward. Se biology has already broadened our knowledge about the genetic code and about protein translation. It now holds great promises also for a better understanding of some key aspects of cancer, inflammation, fertility and prevention of age-associated diseases.

Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP.

The family M1 of Zn-dependent aminopeptidases comprises members of closely related enzymes which are known to be involved in a variety of physiologically important processes. On the basis of two highly conserved peptide motifs, we have identified a new member of this family by PCR amplification and cDNA-library screening. The longest ORF encodes a protein of 930 residues. It contains the HEXXH(X)18E Zn-binding motif and displays high homology to the other M1 family members except for its N-terminus for which a signal sequence of 20 residues can be predicted. This interpretation was supported by expressing fusion proteins formed with green fluorescent protein which localized to intracellular vesicles in COS-7 and BHK cells. Northern-blot analysis revealed ubiquitous expression of a major 3. 1-kb transcript. For enzymatic studies, the complete protein was expressed in Sf 9 insect cells. When aminoacyl beta-naphthylamides were used as substrates, efficient hydrolysis was only observed for Leu (and to a lesser extent Met). The activity was inhibited by chelators of bivalent cations and by other known aminopeptidase inhibitors, but surprisingly puromycin was without effect. This newly identified puromycin-insensitive leucyl-specific aminopeptidase is a signal-sequence-bearing member of family M1 and may be another example of the small subset of substrate-specific peptidases.

Human TRH-degrading ectoenzyme cDNA cloning, functional expression, genomic structure and chromosomal assignment.

Thyrotropin-Releasing Hormone (TRH) is an important extracellular signal substance that acts as a stimulator of hormone secretion from adenohypophyseal target cells and fulfills many criteria for the function of a neuromodulator/neurotransmitter within the central and peripheral nervous systems. The inactivation of TRH-signals is catalysed by a highly specific ectoenzyme. Here, we characterize the human TRH-degrading ectoenzyme (TRH-DE) by primary sequence, functional expression, genomic structure and chromosomal assignment. By screening a cDNA-library constructed from human lung, 5.7 kb of cDNA were identified. The longest open reading frame predicts a type II integral membrane protein of 117 kDa. The extracellular domain contains the HEXXH + E motif that is characteristic of a certain family of Zn-dependent aminopeptidases. Within this family, the sequences of human and rat TRH-DE reveal an unusual high degree of conservation (96% identical residues). Specific enzymatic activity was observed after transfecting COS-7 cells with human TRH-DE cDNA yielding a Km for TRH hydrolysis of 29.7 microM. Northern blot analysis demonstrated a restricted tissue distribution with highest transcript levels in the brain. Using fluorescent in situ hybridization with the cDNA and a genomic lambda clone, respectively, we localized the TRH-DE gene to the long arm of human chromosome 12. Five independent P1 artificial chromosome clones were required to span the complete cDNA sequence and revealed that it is distributed on 19 exons. Interspecies Southern analysis suggests that the gene is present as a single copy in human, monkey, rat, mouse, dog, bovine, rabbit and chicken DNA. All of these data further the notion that the TRH-DE is not an ordinary enzyme but a specific neuropeptidase that has been highly conserved among species.

Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is an important extracellular signal substance that acts as a hypothalamic-releasing factor, which stimulates the release of adenohypophyseal hormones and functions as a neurotransmitter/neuromodulator in the central and peripheral nervous system. The inactivation of TRH after its release is catalyzed by an ectoenzyme localized preferentially on neuronal cells in the brain and on lactotrophic pituitary cells. This enzyme exhibits a very high degree of substrate specificity as well as other unusual properties. The activity of the adenohypophyseal enzyme is stringently controlled by estradiol and thyroid hormones, indicating that this enzyme itself may serve regulatory functions. Fragments of the enzyme isolated from rat or pig brain were generated by enzymatic digestion or cyanogen bromide cleavage, purified by reverse-phase HPLC, and sequenced. PCR amplification and screening of cDNA libraries from rat brain and pituitary led to the identification and isolation of a cDNA that encodes a protein of 1025 amino acids. The analysis of the deduced amino acid sequence was consistent with the identification of the enzyme as a glycosylated, membrane-anchored Zn metallopeptidase. Furthermore, Northern blot analysis demonstrated that the mRNA levels paralleled the tissue distribution of the enzyme and that in pituitary tissue the transcript levels rapidly increased when the animals were treated with triiodothyronine. Finally, transient transfection of COS-7 cells with this cDNA led to the expression of an active ectopeptidase that displayed the characteristics of the TRH-degrading ectoenzyme.