Evidence that a polymorphism within the 3′UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer

Low selenium (Se) status has been associated with increased risk of colorectal cancer (CRC). Se is present as the amino acid selenocysteine in selenoproteins, such as the glutathione peroxidases. Se incorporation requires specific RNA structures in the 3' untranslated region (3'UTR) of the selenoprotein mRNAs. A single nucleotide polymorphism (SNP) occurs at nucleotide 718 (within the 3'UTR) in the glutathione peroxidase 4 gene. In the present study, Caco-2 cells were transfected with constructs in which type 1 iodothyronine deiodinase coding region was linked to the GPx4 3'UTR with either C or T variant at position 718. Higher reporter activity was observed in cells expressing the C variant compared to those expressing the T variant, under either Se-adequate or Se-deficient conditions. In addition, a disease association study was carried out in cohorts of patients with either adenomatous polyps, colorectal adenocarcinomas and in healthy controls. A higher proportion of individuals with CC genotype at the GPx4 T/C 718 SNP was present in the cancer group, but not in the polyp group, compared with the control group (P < 0.05). The present data demonstrate the functionality of the GPx4 T/C 718 SNP and suggest that T genotype is associated with lower risk of CRC.     

Human selenium-dependent thioredoxin reductase from HeLa cells: properties of forms with differing heparin affinities.

The TrxRl form of thioredoxin reductase (TrxR) was the major form of the enzyme isolated from HeLa cells grown in a fermentor at 35 degrees C under controlled aeration conditions favorable to growth, nominally 30% of saturation of dissolved oxygen or 8 ml of oxygen in a liter of medium. This TrxR1 form was not retained on a heparin affinity matrix, it contained one selenium per subunit, was highly active catalytically, and showed strong cross-reactivity with anti-rat liver TrxR1 polyclonal antibodies. At higher aeration, 50% of saturation of dissolved oxygen or 12 ml of oxygen in a liter of medium, HeLa cell growth was slower and additional TrxR forms that bound to heparin were present in purified enzyme preparations. A minor component, TrxR2, the mitochondrial form of TrxR, was detected in the heparin-bound enzyme fraction. One enzyme form that contained less selenium (ca. 0.5 Se per TrxR subunit) was only about 50% as active with thioredoxin or 5,5'dithiobis(2-nitrobenzoic acid) as substrate. Cross-reactivity of this form with anti-rat liver TrxR1 polyclonal antibodies was very weak. The isoelectric point of the low Se enzyme, 5.85, was higher than that, 5.2-5.4, of normal Se content enzyme. Affinity of purified fully active TrxR1 to heparin could be induced by reduction with NADPH or tris-(2-carboxyethyl)phosphine (TCEP). Under anaerobic conditions there was complete retention of Se indicating that an enzyme conformation change effected by reduction was involved. The TCEP-reduced enzyme form was very oxygen labile and upon exposure to air both the Se content and catalytic activity decreased by about 50%. Addition of millimolar concentrations of NADPH or NADP(+) to the TCEP-reduced enzyme gave full protection from oxygen inactivation. TrxR1 exhibited weak peroxidase activity with H(2)O(2) as substrate in the presence of an NADPH-generating system but this activity was unstable. Specific alkylation of the selenocysteine residue of TrxR1 which completely inhibits the NADPH-dependent reduction of disulfides also destroyed peroxidase activity.     

Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells. Effect on cell growth and differentiation

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.     

Effect of sodium selenosulfate on restoring activities of selenium-dependent enzymes and selenium retention compared with sodium selenite in vitro and in vivo

Sodium selenosulfate has been extensively used as a precursor of selenide ions in the preparation of nano Se-containing compounds. Its biological properties remain completely unknown. Sodium selenosulfate and sodium selenite were added to the medium of HepG2 cells and administered intraperitoneally at a dose of 0.1 mg Se/kg body weight to selenium-deficient mice, respectively. Both of the selenium compounds could increase the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) in a dose-dependent manner in cells and efficiently restore selenium retention and activities of GPx and TrxR in mice. All of the variables were in correlation with the Se supply. There was no distinction in elevating activities of GPx and TrxR between selenosulfate and selenite in vitro. After a 2-d supply of selenosulfate, the activity of GPx in the liver was 65% (p < 0.001) and Se accumulations in the liver, kidney and blood were 64%, 86%, and 65%, respectively, of those treated with selenite (allp < 0.01). With the 7-d selenosulfate supplementation, the activity of GPx in the kidney and activities of TrxR in the liver and kidney were 88%, 75%, and 78%, respectively, of those treated with selenite (allp < 0.01); Se retentions in the liver and kidney were 85% and 93%, respectively of those supplemented with selenite (bothp < 0.01). These facts indicated that selenosulfate could be absorbed and utilized in the biological system. No difference in vitro demonstrated that selenosulfate could be absorbed and generate reduced selenide as efficiently as selenite. The differences between the two compounds in vivo were the result of other factors that affected selenosulfate utilization in tissues.     

A luciferase reporter assay to investigate the differential selenium-dependent stability of selenoprotein mRNAs

The mechanisms regulating the differential selenium (Se)-dependent stability of selenoprotein mRNAs are partially characterized. To further study the Se-dependent regulation of selenoproteins, we developed a novel chemiluminescent reporter to monitor the steady-state mRNA level of an artificial selenoprotein. Our reporter is a fusion of the Renilla luciferase gene and of the β-globin gene, but contains features required for incorporation of selenocysteine (SEC), namely, a UGA-SEC codon and a 3' untranslated region RNA stem loop called a SEC incorporation sequence (SECIS). At various levels of Se, the activity of reporters containing GPX1 or GPX4 SECIS elements is proportional to the steady-state mRNA level of the reporter construct and reflects the level of the corresponding endogenous mRNA. In a reporter containing a UGA codon and a functional GPX1 SECIS, Se-dependent nonsense-mediated decay (NMD) occurred in the cytoplasm, as opposed to the more typical nuclear location. To validate the reporter system, we used genetic and pharmacologic approaches to inhibit or promote NMD. Modulation of UPF1 by siRNA, overexpression, or by inhibition of SMG1 altered NMD in this system. Our reporter is derived from a Renilla luciferase reporter gene fused to an intron containing B-globin gene and is subject to degradation by NMD when a stop codon is inserted before the second intron.     

Comparing functional metabolic effects of marginal and sufficient selenium supply in sheep

This study was performed to characterise key data of long-term ovine Se metabolism and to work out the best biomarker of Se status. An upgrade from marginal (<0.05 mg Se/kg diet, ‘Se?’) to sufficient (0.2 mg Se/kg diet, ‘Se+’) nutritional Se supply using sodium selenite was monitered biweekly by analysing Se concentration, glutathione peroxidase (Gpx) activity and routine biochemistry in blood/serum over 2 years. Se, Cu, Zn, cytosolic Gpx and thioredoxin reductase (TrxR) activity were measured in the liver (biopsies/post-mortem). Se, Gpx, TrxR, glutathione-S-transferase-alpha (aGST) and iodothyronine deiodinase (Dio1) were analysed in the kidney, heart muscle and thyroid. Relative mRNA expression of hepatic aGST1 and Gpx1 was determined.Improvement of Se supply strongly increased serum and liver Se concentration within 10 and 20 days, respectively followed by a plateau. Whereas the achievement of a maximum whole blood Gpx activity was reached after 3 months, serum Gpx3 activity increased with high variations. Hepatic Gpx activity reached a maximum during days 100–200, decreasing thereafter. Distinct group differences in Se and cytosolic Gpx activity were evident in all organs (except Se in kidney). TrxR and Dio1 activity was affected only in the liver. The Se? sheep showed an ongoing decrease in serum Se concentration within 2 years, whereas liver Se remained almost unaffected. High relative Gpx1 mRNA expression in the Se+ group was consensual to high hepatic Gpx activity. Relative mRNA expression of hepatic aGST1 was higher in the Se? sheep. Clinical signs and abnormalities in routine biochemistry were absent.In summary, the best biomarker of Se deprivation and nutritional Se upgrade, respectively was Se in serum. Moreover, hepatic Se concentrations reliably reflected the upgrade of Se supply within days. Whole blood Gpx reacts slowly depending on newly formed erythrocytes restricting its diagnostic use. Vital organs are affected by Se deficiency due to a decrease of cytosolic Gpx activity attenuating the antioxidative system. Cellular up-regulation of aGST1 mRNA expression in the Se? group is assumed to partially compensate for the decreased antioxidant defence due to a loss in Gpx activity. This sheep model appears advantageous for long-term studies on sub-clinical metabolic effects in experimental modifiable nutritional Se supply.     

Effect of dietary selenium on the promotion of hepatocarcinogenesis by 3,3', 4,4'-tetrachlorobiphenyl and 2,2', 4,4', 5,5'-hexachlorobiphenyl

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that have promoting activity in the liver. PCBs induce oxidative stress, which may influence carcinogenesis. Epidemiological studies strongly suggest an inverse relationship between dietary selenium (Se) and cancer. Despite evidence linking Se deficiency to hepatocellular carcinoma and liver necrosis, the underlying mechanisms for Se cancer protection in the liver remain to be determined. We examined the effect of dietary Se on the tumor promoting activities of two PCBs congeners, 3,3', 4,4'-tetrachlorobiphenyl (PCB-77) and 2,2', 4,4', 5,5'-hexachlorobiphenyl (PCB-153) using a 2-stage carcinogenesis model. An AIN-93 torula yeast-based purified diet containing 0.02 (deficient), 0.2 (adequate), or 2.0 mg (supplemental) selenium/kg diet was fed to Sprague-Dawley female rats starting ten days after administering a single dose of diethylnitrosamine (150 mg/kg). After being fed the selenium diets for 3 weeks, rats received four i.p. injections of either PCB-77 or PCB-153 (150 micromol/kg) administered every 14 days. The number of placental glutathione S-transferase (PGST)-positive foci per cm(3) and per liver among the PCB-77-treated rats was increased as the Se dietary level increased. Unlike PCB-77, rats receiving PCB-153 did not show the same Se dose-response effect; nevertheless, Se supplementation did not confer protection against foci development. However, the 2.0 ppm Se diet reduced the mean focal volume, indicating a possible protective effect by inhibiting progression of preneoplastic lesions into larger foci. Cell proliferation was not inhibited by Se in the liver of the PCB-treated groups. Se did not prevent the PCB-77-induced decrease of hepatic Se and associated reduction in glutathione peroxidase (GPx) activity. In contrast, thioredoxin reductase (TrxR) activity was not affected by the PCBs treatment or by Se supplementation. These findings indicate that Se does not inhibit the number of PGST-positive foci induced during promotion by PCBs, but that the size of the lesions may be inhibited. The effects of Se on altered hepatic foci do not correlate with its effects on GPx and TrxR.     

Selenoproteins and selenium status in bone physiology and pathology

Background

Emerging evidence supports the view that selenoproteins are essential for maintaining bone health.

Scope of review

The current state of knowledge concerning selenoproteins and Se status in bone physiology and pathology is summarized.

Major conclusions

Antioxidant selenoproteins including glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), as a whole, play a pivotal role in maintaining bone homeostasis and protecting against bone loss. GPx1, a major antioxidant enzyme in osteoclasts, is up-regulated by estrogen, an endogenous inhibitor of osteoclastogenesis. TrxR1 is an immediate early gene in response to 1α,25-dihydroxyvitamin D3, an osteoblastic differentiation agent. The combination of 1α,25-dihydroxyvitamin D3 and Se generates a synergistic elevation of TrxR activity in Se-deficient osteoblasts. Of particular concern, pleiotropic TrxR1 is implicated in promoting NFκB activation. Coincidentally, TrxR inhibitors such as curcumin and gold compounds exhibit potent osteoclastogenesis inhibitory activity. Studies in patients with the mutations of selenocysteine insertion sequence-binding protein 2, a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins have clearly established a causal link of selenoproteins in bone development. Se transport to bone relies on selenoprotein P. Plasma selenoprotein P concentrations have been found to be positively correlated with bone mineral density in elderly women.

General significance

A full understanding of the role and function of selenoproteins and Se status on bone physiology and pathology may lead to effectively prevent against or modify bone diseases by using Se.     

Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines

Background

Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy.

Mercury and selenium interaction in vivo: effects on thioredoxin reductase and glutathione peroxidase

Mercury compounds exert toxic effects via interaction with many vital enzymes involved in antioxidant regulation, such as selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx). Selenium supplementation can reactivate the mercury-inhibited TrxR and recover the cell viability in vitro. To gain an insight on how selenium supplementation affects mercury toxicity in vertebrates, we investigated the effects of selenium on the mercury accumulation and TrxR and GPx activities in a fish model. Juvenile zebra-seabreams were exposed either to methylmercury (MeHg) or inorganic mercury (Hg(2+)) in the presence or absence of sodium selenite (Se) for 28 days followed by 14 days of depuration. Mercury accumulation was found to be 10-fold higher under MeHg exposure than under Hg(2+) exposure. Selenium supplementation caused a half decrease of the accumulation of MeHg but did not influence Hg(2+) accumulation. Exposure to both mercurials led to a decrease of the activity of TrxR (<50% of control) in all organs. Se supplementation coincident with Hg(2+) exposure protected the thioredoxin system in fish liver. However, supplementation of Se during the depuration phase had no effects. The activity of GPx was only affected in the brain of fishes upon the exposure to MeHg and coexposure to MeHg and Se. Selenium supplementation has a limited capacity to prevent mercury effects in brain and kidney. These results demonstrate that Se supplementation plays a protective role in a tissue-specific manner and also highlight the importance of TrxR as a main target for mercurials in vivo.     

Selenium-regulated translation control of heterologous gene expression: Normal function of selenocysteine-substituted gene products

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a “selenium translation element” in the 3′ untranslated region of mRNA. The selenium translation element is required to re-interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium-dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in-frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3′ untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor β1. UGA (opal) mutant-STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium-dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild-type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium-dependent translational control to the expression of a wide variety of target genes, allows facile ⁷⁵Se radioisotopic labeling of the heterologous proteins, and permits site-specific heavy atom substitution. © 1996 Wiley-Liss, Inc.     

Incorporation of selenium from selenite and selenocystine into glutathione peroxidase in the isolated perfused rat liver

The erythrocyte-free, isolated perfused rat liver was used to study the incorporation of selenium into glutathione peroxidase. Gel filtration and ion exchange chromatography of liver supernatant demonstrated ⁷⁵Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced ⁷⁵Se incorporation from L[⁷⁵Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting ⁷⁵Se incorporation from [⁷⁵Se]selenite. These results indicate that selenide and selenite are more readily metabolized than is selenocysteine to the immediate selenium precursor used for glutathione peroxidase synthesis, and suggest a posttranslational modification at another amino acid residue, rather than direct incorporation of selenocysteine, as the mechanism for formation of the presumed selenocysteine moiety of the enzyme.     

谷胱甘肽过氧化物酶的硒代半胱氨酸插入元件

真核生物将硒代半胱氨酸插入蛋白质必需硒代半胱氨酸插入元件（ＳＥＣＩＳ）的参与,后者位于硒蛋白ｍＲＮＡ的３′非翻译区．采用ＲＮＡ折叠程序对１５个谷胱甘肽过氧化物酶基因进行计算机处理发现,其可能的ＳＥＣＩＳ中都具有３段保守碱基ＡＵＧＡ－Ａ（Ｇ）ＡＡ－ＧＡ．根据Ａ（Ｇ）ＡＡ位于顶环或者顶环上游５′臂的突环上,可将ＳＥＣＩＳ分为Ⅰ型和Ⅱ型结构     

cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1).

Cytosolic thioredoxin (Trx) and thioredoxin reductase (TrxR) comprise a ubiquitous system that uses the reducing power of NADPH to act as a general disulfide reductase system as well as a potent antioxidant system. Human and rat mitochondria contain a complete thioredoxin system different from the one present in the cytosol. The mitochondrial system is involved in the oxidative stress protection through a mitochondrial thioredoxin-dependent peroxidase. We report here the cDNA cloning and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene (TrxR2). The mouse TrxR2 cDNA encodes for a putative protein of 527 amino acid residues with a calculated molecular mass of 57 kDa, that displays high homology with the human and rat counterparts. The N-terminus of the protein displays typical features of a mitochondrial targeting sequence with absence of acidic residues and abundance of basic residues. Mouse TrxR2 also contains a stop codon in frame at the C-terminus of the protein, necessary for the incorporation of selenocysteine that is required for enzymatic activity. The typical stem-loop structure (SECIS element) that drives the incorporation of selenocysteine is identified in the 3'-UTR. Northern analysis of the mouse TrxR2 mRNA shows a similar pattern of expression with the human homologue, with higher expression in liver, heart and kidney. Finally, we have assigned the mouse TrxR2 gene to chromosome 16 mapping at 11.2 cM from the centromer and linked to the catechol-o-methyltransferase (comt) gene.     

Chemopreventive activity of selenocysteine prodrugs against tobacco‐derived nitrosamine (NNK) induced lung tumors in the A/J mouse

Prodrugs of L ‐selenocysteine have potential utility in cancer chemoprevention. This study reports the efficacy of three selenazolidine‐4(R)‐carboxylic acids, (2‐unsubstituted, 2‐oxo, and 2‐methyl derivatives; SCA, OSCA, and MSCA, respectively) against tobacco‐related lung tumorigenesis in a mouse model. Seven days after initiation of an AIN‐76A diet supplemented with sodium selenite (5 ppm Se), L ‐selenomethionine (3.75 ppm Se), Se‐methyl‐L ‐selenocysteine (3 ppm Se), L ‐selenocystine (15 ppm Se), SCA (15 ppm Se), OSCA (15 ppm Se), or MSCA (15 ppm Se), mice received 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK; 10 μmol, i.p.). After an additional 16 weeks on the diets, two compounds, OSCA and selenocystine, significantly reduced lung adenoma multiplicity from 7.2 tumors per mouse in the NNK group to 4.5 and 4.6 tumors per mouse, respectively. Neither selenium concentration nor glutathione peroxidase activity in either RBCs or liver served as surrogate indicators of tumor reduction. Hepatic selenium levels were significantly elevated by all selenium‐containing compounds except Se‐methyl‐L ‐selenocysteine and SCA; RBC selenium levels by all except sodium selenite and MSCA. With the exception of L ‐selenomethionine, RBC glutathione peroxidase activity was increased along with the elevated selenium levels. Hepatic glutathione peroxidase activity was elevated by all Se‐compounds except SCA. The two compounds showing significant tumor reduction (OSCA and selenocystine) were the only two compounds that showed ubiquity of changes, elevating both selenium levels and GPx activity in both liver and RBC. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:396‐405, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20105