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Selenoproteins play important roles in many cellular functions and biochemical pathways in mammals. Our previous study showed that the deficiency of the 15 kDa selenoprotein (Selenof) significantly reduced the formation of aberrant crypt foci (ACF) in a mouse model of azoxymethane (AOM)-induced colon carcinogenesis. The objective of this study was to examine the effects of Selenof on inflammatory tumorigenesis, and whether dietary selenium modified these effects. For 20 weeks post-weaning, Selenof-knockout (KO) mice and littermate controls were fed diets that were either deficient, adequate or high in sodium selenite. Colon tumors were induced with AOM and dextran sulfate sodium. Surprisingly, KO mice had drastically fewer ACF but developed a similar number of tumors as their littermate controls. Expression of genes important in inflammatory colorectal cancer and those relevant to epithelial barrier function was assessed, in addition to structural differences via tissue histology. Our findings point to Selenof’s potential role in intestinal barrier integrity and structural changes in glandular and mucin-producing goblet cells in the mucosa and submucosa, which may determine the type of tumor developing.

Summary: Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins. : Yim et al. report that Selenof (15-kDa selenoprotein; Sep15) functions as a gatekeeper of immunoglobulins and, likely, other client proteins en route from the ER to the Golgi apparatus, thereby preventing secretion of dysfunctional proteins and supporting redox quality control. Keywords: Selenof, selenoprotein, oxidoreductase, immunoglobulins, IgM, knockout mouse, endoplasmic reticulum, Sep15, gatekeeper

OBJECTIVES: Plant secondary metabolites, such as polyphenols, are found in many fruits, grains, and vegetables, and are thus a part of normal human diet. One such food is sorghum (Sorghum bicolor), a cereal grain that contains varying concentrations of polyphenols. Many polyphenols have been implicated in the regulation of colon cancer through modulating the antioxidant defense, which includes some of the major selenoproteins, e.g., thioredoxin reductases (TXNRD) and glutathione peroxidases (GPX). However, because such redox-active enzymes have been shown to be involved in both cancer prevention and promotion, the goal of our study is to assess the impact of high-polyphenol sorghum extracts on the expression of selenoproteins. METHODS: Human colon cancer cells (HT29, HCT116) were incubated with 1.25 mg high-polyphenol sorghum bran extract per mL medium for 48 h. RNA was extracted with Trizol/Chloroform, and reverse-transcribed to cDNA. mRNA expression of selenoproteins was quantitated using qPCR, normalized to GAPDH, and analyzed using GraphPad Prism. Protein lysates will be used for Western blotting and catalytic activity assays. RESULTS: Compared to solvent control, incubation of human colon cancer cells with high-polyphenol sorghum extracts for 48 h moderately impacted mRNA expression of investigated selenoproteins. One of the extracts resulted in a nearly doubled GPX1 mRNA expression in HCT116 cells (p = 0.08), whereas preliminary results suggest that TXNRD1 expression may be lowered. CONCLUSIONS: High-polyphenol varieties of sorghum may affect selenoprotein expression. Further investigations involving both shorter and longer-term incubation times, as well as effects on protein expression and activity will help elucidate the effects of these new sorghum varieties on selenoprotein expression important in prevention and promotion of colon cancer. FUNDING SOURCES: Financial support was provided by Towson University's Fisher College of Science and Mathematics (P. Tsuji) and the USDA (D. Smolensky).

The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4−/− mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4−/− livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4−/− pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4−/− mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation., Graphical abstract, Highlights • Conditional Gpx4 loss causes hepatocellular degeneration and early death of mice. • Dietary vitamin E supplementation rescues death of liver-specific Gpx4 null mice. • Nutritional vitamin E content in diet may severely impact experimental outcome.

Selenium is an essential element that is required to support a number of cellular functions and biochemical pathways. The objective of this study was to examine the effects of reduced dietary selenium levels on gene expression to assess changes in expression of non-selenoprotein genes that may contribute to the physiological consequences of selenium deficiency. Mice were fed diets that were either deficient in selenium or supplemented with selenium in the form of sodium selenite for six weeks. Differences in liver mRNA expression and translation were measured using a combination of ribosome profiling, RNA-Seq, microarrays, and qPCR. Expression levels and translation of mRNAs encoding stress-related selenoproteins were shown to be up-regulated by increased selenium status, as were genes involved in inflammation and response to interferon-γ. Changes in serum cytokine levels were measured which confirmed that interferon-γ, as well as IL-6, were increased in selenium adequate mice. Finally, microarray and qPCR analysis of lung tissue demonstrated that the selenium effects on immune function are not limited to liver. These data are consistent with previous reports indicating that adequate selenium levels can support beneficial immune responses, and further identify the IL-6 and interferon-γ pathways as being responsive to dietary selenium intake.

Selenocysteine-containing proteins (selenoproteins) have been implicated in the regulation of various cell signaling pathways, many of which are linked to colorectal malignancies. In this in-depth excurse into the selenoprotein literature, we review possible roles for human selenoproteins in colorectal cancer, focusing on the typical hallmarks of cancer cells and their tumor-enabling characteristics. Human genome studies of single nucleotide polymorphisms in various genes coding for selenoproteins have revealed potential involvement of glutathione peroxidases, thioredoxin reductases, and other proteins. Cell culture studies with targeted down-regulation of selenoproteins and studies utilizing knockout/transgenic animal models have helped elucidate the potential roles of individual selenoproteins in this malignancy. Those selenoproteins, for which strong links to development or progression of colorectal cancer have been described, may be potential future targets for clinical interventions.

The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility.

The selenocysteine (Sec) tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2'-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified posttranscriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA[Ser]Sec population by one or more procedures, and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA[Ser]Sec and resolving the isoforms are used in only a few laboratories, these procedures will be discussed briefly and references provided for more detailed information, while the more recently developed procedures are discussed in detail. In addition, a novel technique that was developed in sequencing tRNA[Ser]Sec for identifying their occurrence in other organisms is also presented.

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.

International audience; The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Selenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic 75Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 ± 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G2-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle. Cancer Prev Res; 3(5); 630–9. ©2010 AACR.

When selenocysteine (Sec) tRNA[Ser]Sec was originally discovered, it was proposed to be the first nonsense suppressor tRNA found in mammalian and avian tissues, since it exclusively decoded the nonsense codon, UGA, which normally dictates the cessation of protein synthesis. This tRNA was subsequently shown to be Sec tRNA, which inserted Sec into protein as the 21st proteinogenic amino acid. Once it was established that this tRNA was aminoacylated with serine by seryl-tRNA synthetase and served as the scaffold for Sec synthesis, Sec tRNA was appropriately named Sec tRNA[Ser]Sec. The mammalian Sec-tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2′-O-methyl moiety on the uridine at position 34, designated Um34. The non-Um34 isoform is involved in the synthesis of a subclass of selenoproteins, called housekeeping selenoproteins, while the Um34 isoform supports synthesis of stress-related selenoproteins. These novel functions and other unique features of Sec tRNA are the subjects of this chapter, supporting the idea that this tRNA is the quintessential constituent in selenoprotein biosynthesis.

Selenium (Se) exerts its mechanistic effects through incorporation into selenoproteins. Many selenoproteins, such as the 15kDa selenoprotein (Sep15), are involved in cancer prevention and promotion...

Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology, and did not activate endoplasmic reticulum stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from an improper folding status of lens proteins caused by Sep15 deficiency.

Selenium, an essential micronutrient, is recognized as a cancer protective agent that functions through low-molecular weight selenocompounds, and, importantly, through selenoproteins. Previous studies suggest that the 15 kDa selenoprotein (Sep15), a gene polymorphic in humans, has an important role in colon cancer in vivo, and that reduction of Sep15 in human HCT116 colon cancer cells reverses their cancer phenotype. Our diet contains a wide array of nutrients, and it is conceivable that these share molecular targets with selenium and regulate selenoprotein expression. The selenoproteins Thioredoxin Reductase 1 (TR1) and Glutathione Peroxidase (GPx) 2 are regulated by the transcription factor Nrf2, whereas GPx1 is not. Some dietary compounds, e.g., Sulforaphane, are known Nrf2-inducers, impacting expression of TR1 and GPx2. However, the effect of Sulforaphane and many polyphenols on other selenoproteins, or, how other polyphenols influence TR1 and GPx2 remains to be elucidated. Using HCT116 cells, we are investigating the effects of polyphenols and Sulforaphane on selenoprotein expression. Cells with wild type or down-regulated expression of Sep15 were exposed to 5 μM Sulforaphane or 20 μM 5,7-Dimethoxyflavone (5,7-DMF) for 48 hours. Subsequently, mRNA was isolated and examined with real-time RT-PCR. Our preliminary results suggest that, Sulforaphane exposure resulted in a slight increase of TR1 mRNA in both control and shSep15 cells, but not GPx2. Interestingly, 5,7-DMF exposure resulted in increased GPx1 (P0.05) mRNA in control cells. Surprisingly, GPx2 mRNA appeared significantly down-regulated (P Citation Format: Sarah E. Galinn, Lindsay C. Rosenthal, Steven M. Barsotti, Petra A. Tsuji. Influence of polyphenols on selenoprotein expression in human colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2013-4881