Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 microgram/ml) and tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-alpha exerted a transient effect on the GPX activity. The treatment with SNAP (200 microM) or sodium nitroprusside (200 microM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-alpha accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

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 microM 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 microM 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.     

Selenium prevents downregulation of antioxidant selenoprotein genes by methylmercury

Selenium (Se) is an essential nutrient required by Se-dependent proteins, termed selenoproteins. The selenoprotein family is small but diverse and includes key proteins in antioxidant, redox signaling, thyroid hormone metabolism, and protein folding pathways. Methylmercury (MeHg) is a toxic environmental contaminant that affects seafood safety. Selenium can reduce MeHg toxicity, but it is unclear how selenoproteins are affected in this interaction. In this study we explored how Se and MeHg interact to affect the mRNA expression of selenoprotein genes in whole zebrafish (Danio rerio) embryos. Embryos were obtained from adult zebrafish fed MeHg with or without elevated Se in a 2×2 factorial design. The embryo mRNA levels of 30 selenoprotein genes were then measured. These genes cover most of the selenoprotein families, including members of the glutathione peroxidase (GPX), thioredoxin reductase, iodothyronine deiodinase, and methionine sulfoxide reductase families, along with selenophosphate synthetase 2 and selenoproteins H, J-P, T, W, sep15, fep15, and fam213aa. GPX enzyme activity and larval locomotor activity were also measured. We found that around one-quarter of the selenoprotein genes were downregulated by elevated MeHg. These downregulated genes were dominated by selenoproteins from antioxidant pathways that are also susceptible to Se-deficiency-induced downregulation. MeHg also decreased GPX activity and induced larval hypoactivity. Elevated Se partially prevented MeHg-induced disruption of selenoprotein gene mRNA levels, GPX activity, and larval locomotor activity. Overall, the MeHg-induced downregulation and subsequent rescue by elevated Se levels of selenogenes regulated by Se status suggest that Se deficiency is a contributing factor to MeHg toxicity.     

Differential effects of selenium and knock-down of glutathione peroxidases on TNFα and flagellin inflammatory responses in gut epithelial cells

Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-κB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-κB response elements and a TATA box or only a TATA box. TNFα and flagellin activated NF-κB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels. GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-κB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent.     

Selenium regulation of thioredoxin reductase activity and mRNA levels in rat liver

Mammalian thioredoxin reductase (TRR; NADPH₂:oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) is a new member of the family of selenocysteine-containing proteins. TRR activity in Se-deficient rat liver is reported to decrease to 4.5 to 15% of the activity in Se-adequate rat liver, similar to the fall in Se-dependent glutathione peroxidase-1 activity. Both glutathione peroxidase-1 enzyme activity and mRNA levels decrease dramatically in Se deficiency, whereas glutathione peroxidase-4 activity only decreases to 40% of Se-adequate levels and mRNA level is little affected by Se deficiency. The purpose of these experiments is to study the effect of Se status on TRR mRNA levels and enzyme activity in our well-characterized rat model, and to compare this regulation directly to the regulation of other Se-dependent proteins in male weanling rats fed Se-deficient diets or supplemented with dietary Se for 28 days. In two experiments, TRR activity in Se-deficient liver decreased to 15% of Se-adequate activity as compared to 2% and 40% of Se-adequate levels for GPX1 and GPX4, respectively. Using ribonuclease protection analysis, we found that TRR mRNA levels in Se-deficient rat liver decreased to 70% of Se-adequate levels. This decrease in TRR mRNA was similar to the GPX4 mRNA decrease in Se-deficient liver in these experiments, whereas GPX1 mRNA levels decreased to 23% of Se-adequate levels. This study clearly shows that TRR represents a third pattern of Se regulation with dramatic down-regulation of enzyme activity in Se deficiency but with only a modest decrease in mRNA level. The conservation of TRR mRNA in Se deficiency suggests that this is a valued enzyme; the loss of TRR activity in Se deficiency may be the cause of some signs of Se deficiency.     

Impacts of glutathione peroxidase-1 knockout on the protection by injected selenium against the pro-oxidant-induced liver aponecrosis and signaling in selenium-deficient mice

Previous research has suggested that repletion of cellular glutathione peroxidase (GPX1) activity by a single injection of Se was dissociated from the Se protection against the pro-oxidant-induced liver necrosis in Se-deficient rodents. Using the GPX1 knockout (GPX1-/-) mice, TUNEL assay, and apoptosis gene expression microarray, we have demonstrated strikingly different impacts of GPX1 knockout on hepatotoxicity and the related signaling induced by an intraperitoneal injection of 12.5 mg paraquat/kg body weight (b.wt.). In both Se-deficient GPX1-/- and wild-type (WT) mice, the paraquat did not induce typical liver necrosis, rather aponecrosis or necrapoptosis, a syncretic process of cell death sharing characteristics of both apoptosis and necrosis. The severity of liver aponecrosis and the associated mortality were reduced to a much greater extent by an injection of Se (ip, 50 microg/kg b.wt. as Na2SeO3) prior to paraquat stress in the WT mice, compared with the GPX1-/- mice. The induced liver aponecrosis seemed to be more apoptotic in the GPX1-/- mice but more necrotic in the WT mice. The paraquat-mediated gene or protein expression of proapoptotic Bax, Bcl-w, and Bcl-X(S), cell survival/death factors GADD45, MDM2, c-Myc, and caspase-3 was upregulated, but that of antiapoptotic Bcl-2 was downregulated in the GPX1-/- mice vs. the WT mice. Overall, these differences between the two groups of mice were related to a low level of liver GPX1 activity in the WT mice that represented < 4% of the normal physiological level. Therefore, the low level of GPX1 activity in the Se-deficient mice can exert a potent role in defending against liver aponecrosis induced by moderate oxidative stress.     

Selenium Deficiency Augments the Levels of Inflammatory Factors and Heat Shock Proteins via the Redox Regulatory Pathway in the Skeletal Muscles of Mice

Dietary selenium (Se) deficiency is known to cause myodynia syndrome and Se influences immune responses by changing the expression of inflammatory cytokines and heat shock proteins (Hsps), but the details are not completely elucidated. In the present study, 72 1-day-old mice were divided into two groups; the first group was fed a Se-sufficient diet, while the second group was fed a Se-deficient diet. Skeletal muscles and blood samples were taken from all mice after 42 days of treatment. The activities of glutathione peroxidase (GPX) and glutathione (GSH), mRNA and protein expression levels of inflammatory cytokines (including TNF-α, inducible NO synthase, cyclooxygenase-2, and prostaglandin E synthases), protein expression levels of NF-κB, and the mRNA expression levels of Hsps in the skeletal muscles of mice were examined. The results showed that GPX and GSH activities were decreased, while the mRNA and protein expression levels of inflammatory cytokines and the mRNA levels of Hsps were increased by Se deficiency in mouse skeletal muscles. In the present study, the protective role of Se in oxidative stress, inflammatory cytokines, and Hsps in the skeletal muscles of mice was summarized.     

Human immunodeficiency virus type 1 Tat protein impairs selenoglutathione peroxidase expression and activity by a mechanism independent of cellular selenium uptake: consequences on cellular resistance to UV-A radiation

The expression of the HIV-1 Tat protein in HeLa cells resulted in a 2.5-fold decrease in the activity of the antioxidant enzyme glutathione peroxidase (GPX). This decrease seemed not to be due to a disturbance in selenium (Se) uptake. Indeed, the intracellular level of Se was similar in parental and tat-transfected cells. A Se enrichment of the medium did not lead to an identical GPX activity in both cell lines, suggesting a disturbance in Se utilization. Total intracellular 75Se selenoproteins were analyzed. Several quantitative differences were observed between parental and tat-transfected cells. Mainly, cytoplasmic glutathione peroxidase and a 15-kDa selenoprotein were decreased in HeLa-tat cells, while phospholipid hydroperoxide glutathione peroxidase and low-molecular-mass selenocompounds were increased. Thioredoxin reductase activity and total levels of 75Se-labeled proteins were not different between the two cell types. The effect of Tat on GPX mRNA levels was also analyzed. Northern blots revealed a threefold decrease in the GPX/glyceraldehyde phosphate dehydrogenase mRNA ratio in HeLa-tat versus wild type cells. By deregulating the intracellular oxidant/antioxidant balance, the Tat protein amplified UV sensitivity. The LD50 for ultraviolet radiation A was 90 J/cm2 for HeLa cells and only 65 J/cm2 for HeLa-tat cells. The oxidative stress occurring in the Tat-expressing cells and demonstrated by the diminished ratio of reduced glutathione/oxidized glutathione was not correlated with the intracellular metal content. Cellular iron and copper levels were significantly decreased in HeLa-tat cells. All these disturbances, as well as the previously described decrease in Mn superoxide dismutase activity, are part of the viral strategy to modify the redox potential of cells and may have important consequences for patients.     

Characterization and expression analysis of seven selenoprotein genes in yellow catfish Pelteobagrus fulvidraco to dietary selenium levels

BackgroundSelenium (Se) appears in the selenoproteins in the form of selenocysteine (Sec) and is important for the growth and development of vertebrates. The present study characterized seven selenoproteins, consisting of the GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3 cDNAs in various tissues of yellow catfish, explored their regulation to dietary Se addition.Methods3′ and 5′ RACE PCR were used to clone full-length cDNA sequences of seven selenoprotein genes (GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3). Their molecular characterizations were analyzed, including conservative motifs and the SECIS elements. The phylogenetic trees were generated through neighbor-joining (NJ) method with MEGA 6.0 with 1000 bootstrap replications. Quantitative real-time PCR was used to explore their mRNA tissue distribution in the heart, anterior intestine, dorsal muscle, head kidney, gill, liver, brain, spleen and mesenteric fat. Yellow catfish (mixed sex) were fed diets with dietary Se contents at 0.03 (low Se), 0.25 (adequate Se) and 6.39 (high Se) mg Se/kg, respectively, for 12 weeks, and their spleen, kidney, testis and brain were used for the determination of the mRNA levels of the seven selenoproteins.ResultsThe seven selenoproteins had similar domains to their corresponding members of other vertebrates. They were widely expressed in nine tissues, including heart, liver, brain, spleen, head kidney, dorsal muscle, mesenteric fat, anterior intestine and gill, but showed tissue-dependent expression patterns. Dietary Se addition affected the expression of the seven genes in spleen, kidney, testis and brain tissues of yellow catfish.ConclusionTaken together, our study demonstrated the characterization, expression and regulation of seven selenoproteins, which increased our understanding of the biological functions of Se and selenoproteins in fish.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 μg/ml) and tumor necrosis factor-α (TNF-α, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-α exerted a transient effect on the GPX activity. The treatment with SNAP (200 μM) or sodium nitroprusside (200 μM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-α, and aminoguanidine blocked the effect. In cells metabolically labeled with ⁷⁵Se, LPS + TNF-α accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Lethal damage to murine L1210 cells by exogenous lipid hydroperoxides: protective role of glutathione-dependent selenoperoxidases

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(-) cells] were less than 10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(-/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(-) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(-) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(-) being much more so (LD50 approximately 10 microM) than Se(+) (LD50 approximately 75 microM). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(-) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.     

Glutathione peroxidase-1 gene knockout on body antioxidant defense in mice

To determine the in vivo role of cellular glutathione peroxidase (E.C.1.11.1.9, GPX1), we challenged the GPX1 knockout [GPX1(-/-)], the GPX1 overexpressing [GPX1(+)], and their respective wild-type (WT) mice of different Se and vitamin E status with acute oxidative stress. After these mice were injected with pro-oxidants paraquat or diquat at 12 to 125 mg/kg of body weight, their survival rate and time were a function of their GPX1 activity levels. The GPX1 protection was associated with attenuation of NADPH and NADH oxidation, protein carbonyl and F(2)-isoprostanes formation, and alanine transaminase release in various tissues, and was irreplaceable by high levels of dietary vitamin E or other selenoproteins. The GPX1 expression was also protective against moderate oxidative stress induced by low levels of paraquat or diquat, particularly in the Se-deficient mice. Alteration of GPX1 expression showed no impact on the expression of other selenoproteins and antioxidant enzymes in unstressed mice. Total Se content in liver of the Se-adequate GPX1(-/-) mice was reduced by 60% the WT controls. In conclusion, normal expression of GPX1 is essential and overexpression of GPX1 is beneficial to protect mice against acute oxidative stress.     

Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.     

Blood selenium and glutathione peroxidase activity of populations in New Zealand, Oregon, and South Dakota

The relationship of whole blood selenium (Se) to glutathione peroxidase (GPX) activity was examined for individuals in New Zealand, Oregon, and South Dakota who represented, respectively, populations with exposure to low, medium, and high amounts of Se. The mean (respective) blood Se levels were 60, 200, and 400 ng/ml. Intergroup differences in blood Se levels were highly significant (P less than 0.001). GPX assays were performed using two variations of an enzyme-coupled procedure to assess the equivalence of the two methods. Despite a fourfold difference in absolute activities measured by these methods, the GPX activities were highly correlated (r = .86) between procedures. Average blood GPX activity was significantly lower (P less than 0.001) for the New Zealand group compared with the other two groups, but there was no difference in GPX activities between the Oregon and South Dakota groups. Linear regression of GPX vs. Se values within each group indicated a significant correlation of these parameters only in the New Zealand group (r = .46, P less than 0.01). Comparison of these parameters for combined data from all three groups also showed a significant positive correlation (r = .60, P less than 0.001). A saturation model (In GPX = k1 + k2 (Se)-1)) fits the combined data better (r = .80, P less than 0.01) than does direct comparison of the two parameters. These results suggest that GPX activity is an appropriate indicator of human Se status only in populations with below normal exposure to Se, as activity of this enzyme is saturated at relatively low levels.     

Modulation of thioredoxin reductase-2 expression in EAhy926 cells: Implications for endothelial selenoprotein hierarchy

Background

We examined the expression of the mitochondrial selenoenzyme TrxR2 in the endothelial cell line EAhy926 under conditions known to modify its cytoplasmic counterpart TrxR1.

Methods

Cells were cultured with varying concentrations of selenite, sulforaphane or the Ca²⁺ ionophore A23187 for 72-h, prior to assay of TrxR concentration and activity. Further cultures underwent prolonged (7-day) Se-depletion before selenoprotein measurement.

Results

In Se-deficient cultures, neither Se, A23187 or sulforaphane affected TrxR2 concentration, while these treatments induced TrxR1 concentration (p < 0.05). When co-incubated, optimal concentrations of Se (40 nM) and sulforaphane (4 μM) only modestly increased TrxR2 protein (∼ 1.3-fold), compared with TrxR1 (∼ 4-fold). In Se-deficient cells, TrxR activity was unaffected by sulforaphane or A23187. Prolonged Se-depletion caused a comparatively small reduction in TrxR2 (66% TrxR2 retained) against TrxR1 and glutathione peroxidase-1 activity (38% and 17% retained, respectively).

Conclusions

The relative resistance of TrxR2 to Se-deprivation and induction by sulforaphane and A23187 suggests TrxR2 lies near the top of the selenoprotein hierarchy in EAhy926 cells and exhibits near maximum expression under a range of culture conditions. In Se deficiency an inactive (possibly truncated) TrxR1 is produced in response to stimulus by sulforaphane and A23187.

General significance

These observations underpin a likely critical antioxidant role for TrxR2 and TrxR1 in the endothelium.     

Effects of selenium and serum on selenoprotein W in cultured L8 muscle cells

When rat L8 muscle cells were cultured to examine the effects of serum and selenium concentration on selenoprotein W levels and glutathione peroxidase (GPX) activities, no significant differences (P > 0.05) were found in selenoprotein W levels and GPX activities during differentiation. With three different forms of selenium, selenoprotein W levels and GPX activities were shown to increase in L8 myotubes cultured in media with these selenocompounds. Selenite was utilized more efficiently than selenocysteine for both selenoprotein W and GPX activity, but selenium as selenomethionine was less available. Both the protein content and mRNA levels for selenoprotein W were affected by the selenium content of the media. Northern blot data indicated that the expression of selenoprotein W mRNA increased significantly when L8 myotubes were cultured with selenium (P > 0.05). L8 myotubes cultured in 10% calf serum (CS) versus 2% CS with or without addition of 10 m selenium indicated that the increase of selenoprotein W level in L8 myotubes cultured with higher serum concentration (10% CS) is due to the higher selenium concentration in media rather than serum itself.     

Glutathione peroxidase activity and selenoprotein W levels in different brain regions of selenium-depleted rats(1)

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.