Selenoprotein Research Articles

Genetic variants in selenoprotein genes increase risk of colorectal cancer

Low selenium (Se) status correlates with increased risk of colorectal cancer (CRC). Since Se exerts its biological roles through the selenoproteins, genetic variations in selenoprotein genes may influence susceptibility to CRC. This study analysed 12 single-nucleotide polymorphisms (SNPs) in selenoprotein genes [glutathione peroxidase 1 (GPX1), GPX4, 15 kDa selenoprotein (SEP15), selenoprotein S (SELS), selenoprotein P (SEPP1) and thioredoxin reductase 2 (TXNRD2)] and in genes that code for a key protein in Se incorporation [SECIS-binding protein 2 (SBP2)] and in antioxidant defence [superoxide dismutase 2 (SOD2)] in relation to sporadic CRC incidence. CRC patients (832) and controls (705) from the Czech Republic were genotyped using allele specific PCR. Logistic regression analysis showed that three SNPs were significantly associated with an altered risk of CRC: rs7579 (SEPP1), rs713041 (GPX4) and rs34713741 (SELS). The association of these SNPs with disease risk remained after data stratification for diagnosis and adjustments for lifestyle factors and sex. Significant two-loci interactions were observed between rs4880 (SOD2), rs713041 (GPX4) and rs960531 (TXNRD2) and between SEPP1 and either SEP15 or GPX4. The results indicate that SNPs in SEPP1, GPX4 and SELS influence risk of CRC. We hypothesize that the two-loci interactions reflect functional interactions between the gene products. We propose that these variants play a role in cancer development and represent potential biomarkers of CRC risk.

Compound deficiency of Nrf2 and selenoproteins in hematopoietic cells leads to severe defects in erythroid and lymphoid lineage development

Abstract 1993 Poster Board I-1015 Precise regulation of reactive oxygen species (ROS) level is essential for normal cellular homeostasis. Defect of ROS scavenging system results in excess accumulation of ROS in cells, leading to the cellular aging and senescence. Nrf2 (NF-E2-related factor 2) is a basic leucine zipper-type transcription factor, which regulates expression of a set of genes related to the ROS scavenging and detoxification. While accumulating lines of evidence support the significance of Nrf2 for the cellular protection in many types of cells, Nrf2-deficient mice live out their natural life span under the homeostatic condition, and show no significant hematological abnormality except for anemia with aging. We previously found that the Nrf2 activity rose in the macrophages and liver cells derived from conditional knockout (CKO) mice of the selenocystein tRNA ( Trsp ) gene. The Trsp gene is essential for production of selenoproteins, including glutathione peroxidase and thioredoxin reductase. Thus, there are two prevalent ROS scavenging systems based on different molecular mechanisms; Nrf2 and selenoproteins seemingly act compensatory and cooperatively for maintenance of hematopoietic homeostasis. To explore the roles Nrf2 and selenoproteins play in hematopoietic cells, we conducted conditional knockout of the Trsp gene by crossing Trsp -floxed mice with the interferon-inducible Mx1-Cre transgenic mice in combination with Nrf2 gene knockout (KO) in germline. The Trsp -CKO mice in the background of Nrf2 wild type ( Trsp single KO mice) showed thymic atrophy with massive reduction of CD4/CD8 double-positive cells in thymus. This T-lymphocytic phenotype was not changed in the Trsp -CKO mice under the circumstance of Nrf2-null condition ( Trsp:Nrf2 double KO mice), suggesting that selenoproteins were prerequisite for the T-cell development. The Trsp single KO mice suffered from mild anemia with increased number of erythroid progenitors in bone marrow and spleen, and importantly this erythroid phenotype was dramatically exacerbated in the Trsp:Nrf2 double KO mice. A large number of poikilocytes and teardrop-shaped cells were observed in the blood smear of the Trsp:Nrf2 double KO mice, whereas these deformed red blood cells were vaguely found in the Trsp single KO mice. Intracellular ROS level was significantly elevated in erythroid progenitors recovered from the Trsp:Nrf2 double KO mice, although that of the Trsp single KO mice were maintained within normal level, indicating that Nrf2 compensates for the defect of ROS scavenging function caused by the loss of selenoproteins in erythroid cells. These results thus demonstrate for the first time that the ROS scavenging conducted by Nrf2 and selenoproteins is essential for the homeostatic maintenance in erythroid and lymphoid cells in a lineage-specific manner. Disclosures: No relevant conflicts of interest to declare.

The role of plasma cytokine levels, CRP and Selenoprotein S gene variation in OA

Investigating the association between plasma levels of cytokines and chemokines, Selenoprotein S (SELS) gene variation and osteoarthritis (OA) subtypes. The genetics of osteoarthritis and progression (GARP) study consists of 191 sibling pairs with symptomatic OA at multiple joint sites. We have measured plasma levels of 17 cytokines and chemokines and genetic variation at the SELS gene. Nine out of 17 serum markers could be assessed quantitatively, whereas eight markers were assessed qualitatively. Principal component analysis (PCA) on the quantitatively assessed markers and serum high sensitive C-reactive protein (S-HsCRP) revealed that three components underlie 61% of the total plasma variation. Three single nucleotide polymorphisms (SNPs) in the SELS gene revealed four common haplotypes, one of which, GAG (frequency 3.5%) showed significant association to an anti-inflammatory (P=0.019) and acute phase related (P=0.036) component. OA subtype analysis showed that one component (mainly representing chemokine variation) was significantly associated to hand OA and disc degeneration (P=0.029 and P=0.010 respectively) as well as a physical component score (PCS) (P=0.042). The CRP related component also showed a strong association to the PCS (P=0.007). SELS haplotypes showed no association to OA subtypes in the GARP study. Genetic variation in the SELS gene associates to components representing inflammatory signaling. Another component, representing chemokine variation, showed association to hand OA and disc degeneration in the GARP study indicating chemokines may contribute to OA pathogenesis.

Evidence of epistasis between interleukin 1 and selenoprotein-S with susceptibility to rheumatoid arthritis

Objective:Selenoprotein-S (SELS) is involved in the stress response within the endoplasmic reticulum (ER) and inflammation. Recently, promoter variants in the SELS gene were shown to be associated with plasma levels...

The role of the selenoprotein S (SELS) gene -105G>A promoter polymorphism in inflammatory bowel disease and regulation of SELS gene expression in intestinal inflammation

Background/aim: Recently, a -105G>A promoter polymorphism coding for selenoprotein S (SELS) has been shown to increase proinflammatory cytokine expression (Nature Genetics 2005; 37:1234–41). We therefore analyzed SELS expression and potential phenotypic consequences of the -105G>A polymorphism in patients with inflammatory bowel disease (IBD).

The role of the selenoprotein S (SELS) gene −105G>A promoter polymorphism in inflammatory bowel disease and regulation of SELS gene expression in intestinal inflammation

Recently, a -105G>A promoter polymorphism coding for selenoprotein S (SELS) has been shown to increase proinflammatory cytokine expression. We, therefore, analyzed SELS expression and potential phenotypic consequences of the -105G>A polymorphism in patients with inflammatory bowel disease (IBD). SELS mRNA was measured by quantitative polymerase chain reaction (PCR) in intestinal epithelial cells (IEC) after stimulation with proinflammatory cytokines and in human colonic biopsies of IBD patients as well as in murine models of ileitis and murine cytomegalovirus (MCMV) colitis. Genomic DNA from 563 individuals (Crohn's disease: n = 205; ulcerative colitis: n = 154; controls: n = 204) was analyzed for the presence of the SELS-105G>A polymorphism and the three nucleotide-binding oligomerization domain-containing protein 2 (NOD2)/caspase recruitment domain-containing protein 15 (CARD15) variants p.Arg702Trp, p.Gly908Arg and p.Leu1007fsX1008. SELS mRNA expression was increased in IEC after stimulation with proinflammatory cytokines, while its expression was not significantly altered in murine ileitis and MCMV colitis and in inflamed ileal and colonic lesions in IBD patients compared with normal controls. The SELS-105G>A polymorphism was observed with similar frequencies in IBD patients and controls and was not associated with a certain disease phenotype or serum tumor necrosis factor alpha (TNF-alpha) levels in these patients. Medium serum TNF-alpha was 1.27 pg/ml in IBD patients, while none of the controls had TNF-alpha concentrations above the detection threshold (P < 0.0001). SELS mRNA expression is upregulated by proinflammatory cytokines in IECs but the SELS-105G>A polymorphism is not associated with IBD susceptibility and does not contribute to a certain disease phenotype or increased TNF-alpha levels in IBD patients.

Selenoprotein deficiency adversely affects T cell development, proliferation, TCR signaling and cell mediated immunity

Selenium is known to enhance immune function, but its mechanism of action is not known. To elucidate the role of selenoproteins (SPs) in T cell immunity, we used loxP-Cre technology to specifically target the removal of the selenocysteine (Sec) tRNA gene (Δtrsp). Flow cytometric analysis in Δtrsp mice revealed that maturation of CD4+ and CD8+ T cells in thymus were reduced by 50% (p<0.0001). In spleen and lymph nodes, the CD8+ T cell population was reduced by approximately 45%. In vitro stimulation of Δtrsp T cells with anti-CD3/CD28 had no effect on IL-2 production, but cell proliferation was reduced by 98% (p<0.0001) and they also underwent greater apoptosis. Furthermore, Δtrsp T cells exhibit a reduced IL2-R expression and defective phosphorylation of p44/p42 MAPK which is involved in signaling cell proliferation. IL-4 and IL-10 production was reduced by 75% (p<0.05, p<0.01, respectively) indicating that the lack of SPs in T cell may impair Th2 immunity. NP-OVA immunized Δtrsp mice have reduced levels of serum Ig. Findings thus far suggest that SPs are essential for normal T cell development, TCR signaling and T cell dependent immunity. We predict and are investigating: 1) SPs through their redox potential properties protect T cells from ROS generated during signaling events of immune response, and 2) the Δtrsp T cell mice are immunocompromised. This research was supported by the Intramural Research Program of the NIH, NCI.

A comparative analysis of selenoproteins and global gene expression in liver selenocysteine tRNA knockout mice and its rescued variants

Selenium (Se) is an essential micronutrient for mammals, being incorporated into selenoproteins (SPs) as the 21st amino acid, selenocysteine (Sec). Rodents and humans encode 24 and 25 SP genes, respectively. SPs are tissue specific, performing biologically diverse functions. Sec expression is dependent on Sec tRNA, thus, manipulating the expression of Sec tRNA can alter SP expression. Targeted removal of the Sec tRNA gene (trsp) in liver demonstrated the significance of SPs in liver function (Carlson et al., J. Biol. Chem., 2004, 279:). The SP population was replaced in these mice with mutant variants of trsp (A34 and G37) and microarrays were used for comparative analysis of gene expression in liver of knockout and rescued mice. The SPs in these mice were further assessed through real-time PCR. Studies revealed a loss of expression of most SPs in the knockout mice with an enhanced expression of some detoxification genes. However, in the replacement mice, most SPs were expressed similar to the wild type with a corresponding decrease in the expression of detoxification genes as compared to the knockout. Taken together, the data suggest a relationship between SPs and detoxification processes, wherein some specific SPs may be compensated for by the upregulation of genes involved in detoxification in liver of the knockout mouse. This research was supported by the Intramural Research Program of the NIH, NCI, CCR, and by NIH GM061603.

Both selenoproteins and low molecular weight selenocompounds reduce colon cancer risk in mice with genetically impaired selenoprotein expression

It is not known whether selenoproteins (SPs) and/or low molecular weight (MW) selenocompounds are responsible for the cancer protective effects of selenium (Se). Herein, transgenic mice were used that carry a mutant selenocysteine (Sec) tRNA[Ser]Sec transgene which causes reduced SP synthesis in order to evaluate the contribution of SPs to this process. Se homeostasis was characterized in liver and colon of wild type and transgenic mice fed Se deficient diets supplemented with 0, 0.1, or 2.0 μg Se (as selenite)/g diet. 75Se-labeling, western blot analysis, and enzymatic activities revealed that transgenic mice have significantly (p<0.05) reduced liver and colon glutathione peroxidase, but conserved thioredoxin reductase, regardless of Se status. Transgenic mice had signficantly (p<0.01) more Se in the low MW fraction of liver and colon than wild type mice. Compared to wild type mice, transgenic mice had significantly (p<0.05) more azoxymethane-induced aberrant crypt formation (a preneoplastic lesion for colon cancer). Supplemental Se significantly decreased (p<0.05) the number of aberrant crypts and aberrant crypt foci in both mouse strains. These results provide evidence that a lack of SP activity increases colon cancer susceptibility, and that low MW selenocompounds reduce preneoplastic lesions independent of SP genotype. Therefore, both SPs and low MW selenocompounds are important for the cancer protective effects of Se. This research was supported by the Division of Cancer Prevention and the Intramural Research Program of the NIH, NCI.

The selenocysteine tRNA Staf region is essential for adequate selenoprotein levels and mouse longevity

Selenocysteine (Sec) tRNA has been described as the key molecule and the central component in selenoprotein (SP) biosynthesis and is the only known tRNA that governs the expression of an entire class of proteins, the SPs. This provides a unique opportunity to study the expression of SPs by manipulating the levels and characteristics of Sec tRNA. A cDNA encoding a protein that binds to the activator element of Sec tRNA was cloned from X. laevis and named Staf (for Sec tRNA gene transcription activating factor) (Adachi, et al. J Biol Chem 273:, 1998). Herein, we generated a transgenic mouse harboring multiple copies of a transgene carrying a 14bp deletion in the Staf binding region of the Sec tRNA gene (trsp), which is located approximately −200bp upstream of trsp. Transgenic mice were bred with a trsp knockout mouse to remove endogenous wild type trsp. Mice dependent on the mutant transgene showed ~80% decrease in the levels of Sec tRNA in liver. Se75-labeling and western blot analysis revealed that several SPs were affected by this mutation to varying degrees depending on tissue type. The mutant mice showed decreased size and body weight and had a lifespan of less than 30 days. Thus, the Staf binding region of trsp is essential for proper amounts of Sec tRNA transcription. These and other recent findings will be presented. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.

Deletion analysis of the rodent selenoprotein W promoter

To identify regulatory elements in the rat selenoprotein W (SeW) promoter, 2090, 1265, 741, and 404 base pair truncations of genomic DNA lying immediately upstream of the SeW coding sequence were cloned into a luciferase reporter vector (pGL3-Basic from Promega, Madison, WI, USA). 3656 and 406 base pair mouse SeW promoter constructs were also compared. SeW promoter activity was assayed in two rat cell lines: L8 muscle cells and C6 brain cells. The SeW promoter was 2–7 times more active ( p < 0.01) than SV40 promoter. Promoter activity of constructs of the SeW promoter ranging from 200 base pairs to 51 base pairs gradually decreased to zero in brain cells, but fell precipitously to zero in muscle cells. Some truncations stimulated promoter activity, suggesting the full-length promoter may contain binding sites for factors that suppress SeW expression.

Selenoprotein W as molecular target of methylmercury in human neuronal cells is down-regulated by GSH depletion

Methylmercury (MeHg) is well known as a neurotoxic chemical. However, little is mentioned about its neurotoxic mechanism or molecular target in human neuronal cells in particular. We show in this study that exposure of human neuronal cell line, SH-SY5Y, to MeHg dose- and time-dependently impairs viability and mRNA expression of selenoprotein W (SeW) with a significant difference, unlike other selenoenzymes such as, SeP, GPX4, 5DI, and 5′DI. Using real-time RT PCR, the influence of selenium (Se) and glutathione (GSH) on SeW expression was also investigated. While Se depletion caused a weakly reduced SeW mRNA levels, additional Se caused an increase of SeW mRNA levels. Although 2 mM GSH had induced a weak shift on SeW level, the expression of SeW mRNA was down-regulated in SH-SY5Y cells treated with 25 μM BSO, an inhibitor of GSH synthesis. To understand the relationship between a decrease of SeW expression and intracellular GSH and ROS, we measured the concentration of intracellular GSH and ROS in cells treated to 1.4 μM MeHg using fluorescence based assays. A positive correlation was found between SeW mRNA level and intracellular GSH but no significant correlation was observed between intracellular ROS and SeW mRNA level or intracellular GSH contents. Therefore, we suggest that SeW is the novel molecular target of MeHg in human neuronal cells and down-regulation of this selenoenzyme by MeHg is dependent not on generation of ROS but on depletion of GSH.

Identification of putative transcription factor binding sites in rodent selenoprotein W promoter

To understand transcriptional regulation of the selenoprotein W (SeW) gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter and antibodies, we observed that specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as to the metal response element (MRE). Although competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore, the SeW gene may be a target for Sp1 whose binding to various regulatory sequences of the SeW promoter may activate or repress the transcription of SeW. The MRE, GRE, AP-1 and LF-A1 sites were also tested but no evidence was obtained for specific binding as indicated by lack of competition with unlabeled probes.

Metamorphosis of the selenoprotein PHGPx during spermatogenesis.

Metamorphosis of the selenoprotein PHGPx during spermatogenesis.

Effect of copper, zinc and cadmium on the promoter of selenoprotein W in glial and myoblast cells

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404 and 1265 bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured C6 (rat glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265 bp (not 404 bp) construct ( p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter.

Selenoprotein W is a glutathione-dependent antioxidant in vivo

The function of selenoprotein W (Se-W) was investigated by cloning the corresponding cDNA from mouse brain and expressing it in CHO cells and H1299 human lung cancer cells. Overexpression of Se-W markedly reduced the sensitivity of both cell lines to H 2O 2 cytotoxicity. The intracellular peroxide concentration of the transfected cells was lower than that of the parental cells in the absence or presence of extracellular H 2O 2. The resistance to oxidative stress conferred by Se-W was dependent on glutathione. Expression of Se-W mutants in which selenocysteine-13 or cysteine-37 was replaced by serine did not confer resistance to H 2O 2, implicating these residues in the antioxidant activity of Se-W in vivo.

Selenoprotein W in overexpressed and underexpressed rat glial cells in culture

Selenium deficiency results in undetectable levels of selenoprotein W (SeW) in muscle but has very little effect upon its content in the brain and thus rat glial cells were studied. Previous work showed that glutathione (GSH) is bound to SeW and this study was undertaken to elucidate its possible antioxidant functions. Full length cDNA of SeW was cloned to inducible LacSwitch expression vector and stably transfected in C6 rat glial cells. After induction, SeW and its mRNA were expressed 22- and 11-fold higher respectively than control. The cDNA coding region of SeW was cloned to the vector in the antisense direction and stably transfected in C6 cells for underexpression of the protein. After induction, SeW expression was reduced to 20% of the control cells. Glutathione peroxidase activity and GSH levels were not significantly different between induced and control cells. There was a greater survival rate of overexpressed than control cells when incubated with 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH), suggesting SeW possibly has an antioxidant function.

Glutathione peroxidase activity and selenoprotein W levels in different brain regions of selenium-depleted rats

Previous studies in selenium (Se)-depleted sheep and rats showed that selenoprotein W (SeW) levels decreased in all tissues except brain. To further investigate this depletion in different parts of the brain, second generation Se-depleted rats were used. Dams consumed a Se-deficient basal diet during gestation and lactation, and deficient rats were obtained by continuation on the same diet. Control rats were fed a diet with 0.1-mg Se/kg diet after weaning. Glutathione peroxidase (GPX) activities were measured for comparative purposes to SeW levels. GPX activity in muscle, skin, spleen, and testis increased about 4-fold with Se repletion and reached a plateau after 6 or 10 weeks, but GPX activity decreased to almost one tenth of the original activity with continuous Se depletion. In contrast, GPX activities increased, rather than declined, in various brain regions (cortex, cerebellum, and thalamus) with time of feeding the deficient diet. An experiment with first generation rats, however, indicated that GPX activity was significantly lower in these three brain regions from rats fed the deficient diet as compared to rats fed the supplemented diet. SeW levels in skin, spleen, muscle, and testis were undetectable in weanling rats, but became detectable after 6 weeks of Se repletion. In contrast, the expression of SeW in cortex, cerebellum, and thalamus was not significantly affected by Se depletion, but increased SeW levels occurred only in thalamus with Se supplementation. The results with GPX using first and second generation rats suggest that there are “mobile” and “immobile” GPX fractions in the brain.

SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy.

Selenocysteine incorporation at UGA codons requires cis-acting mRNA secondary structures and several specialized trans-acting factors. The latter include a selenocysteine-specific tRNA, an elongation factor specific for this tRNA and a SECIS-binding protein, SBP2, which recruits the elongation factor to the selenoprotein mRNA. Overexpression of selenoprotein mRNAs in transfected cells results in inefficient selenocysteine incorporation due to limitation of one or more of these factors. Using a transfection-based competition assay employing overexpression of selenoprotein mRNAs to compete for selenoprotein synthesis, we investigated the ability of the trans-acting factors to overcome competition and restore selenocysteine incorporation. We report that co-expression of SBP2 overcomes the limitation produced by selenoprotein mRNA overexpression, whereas selenocysteyl-tRNA and the selenocysteine-specific elongation factor do not. Competition studies indicate that once bound to SECIS elements, SBP2 does not readily exchange between them. Finally, we show that SBP2 preferentially stimulates incorporation directed by the seleno protein P and phospholipid hydroperoxide glutathione peroxidase SECIS elements over those of other selenoproteins. The mechanistic implications of these findings for the hierarchy of selenoprotein synthesis and nonsense-mediated decay are discussed.

Selenoprotein W: a review.

Purification of selenoprotein W (Se-W) from rat and monkey muscles was shown to exist in multiple forms: with or without reduced glutathione and/or a 41-Da moiety (identity still unknown). TGA is located at coding position 13 in Se-W complementary DNA (cDNA) from all five species studied (rats, mice, sheep, human and monkey). TGA is also the stop codon in the rodents and sheep cDNA, but TAA is the stop codon in primates. There is an 80% homology of the nucleotide sequence in the coding region among the five species of animals, and the predicted amino acid sequences are 83% identical (rodents identical and primates identical). Se-W levels are highest in muscle, heart and brain from sheep and primates, but very low in rodent hearts. Studies with tissue cultures of muscle and brain cells indicated that selenium influenced Se-W levels. Although the metabolic function of Se-W is unknown, preliminary data suggest that it has an antioxidant function.