Selenoprotein W engages in overactive osteoclast differentiation in multiple myeloma.

BACKGROUND: Patients with multiple myeloma exhibit malignant osteolytic bone disease due to excessive osteoclast formation and function. We recently identified that osteoclastogenic stimulator selenoprotein W (SELENOW) is upregulated via ERK signaling and downregulated via p38 signaling during receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation. In the intrinsic physiological process, RANKL-induced downregulation of SELENOW maintains proper osteoclast differentiation; in contrast, forced overexpression of SELENOW leads to overactive osteoclast formation and function. METHODS AND RESULTS: We observed that SELENOW is highly expressed in multiple myeloma-derived peripheral blood mononuclear cells (PBMCs) and mature osteoclasts when compared to healthy controls. Also, the level of tumor necrosis factor alpha (TNFα), a pathological osteoclastogenic factor, is increased in the PBMCs and serum of patients with multiple myeloma. ERK activation by TNFα was more marked and sustained than that by RANKL, allowing SELENOW upregulation. Excessive expression of SELENOW in osteoclast progenitors and mature osteoclasts derived from multiple myeloma facilitated efficient nuclear translocation of osteoclastogenic transcription factors NF-κB and NFATc1, which are favorable for osteoclast formation. CONCLUSION: Our findings suggest a possibility that feedforward signaling of osteoclastogenic SELENOW by TNFα derived from multiple myeloma induces overactive osteoclast differentiation, leading to bone loss during multiple myeloma.

Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation.

Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow-/- BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow-/- BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow-/- BMDMs. Furthermore, Selenow-/- BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.

Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts.

Selenoproteins containing selenium in the form of selenocysteine are critical for bone remodeling. However, their underlying mechanism of action is not fully understood. Herein, we report the identification of selenoprotein W (SELENOW) through large-scale mRNA profiling of receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation, as a protein that is downregulated via RANKL/RANK/tumour necrosis factor receptor-associated factor 6/p38 signaling. RNA-sequencing analysis revealed that SELENOW regulates osteoclastogenic genes. SELENOW overexpression enhances osteoclastogenesis in vitro via nuclear translocation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 mediated by 14-3-3γ, whereas its deficiency suppresses osteoclast formation. SELENOW-deficient and SELENOW-overexpressing mice exhibit high bone mass phenotype and osteoporosis, respectively. Ectopic SELENOW expression stimulates cell-cell fusion critical for osteoclast maturation as well as bone resorption. Thus, RANKL-dependent repression of SELENOW regulates osteoclast differentiation and blocks osteoporosis caused by overactive osteoclasts. These findings demonstrate a biological link between selenium and bone metabolism.

S-Glutathionylation of mouse selenoprotein W prevents oxidative stress-induced cell death by blocking the formation of an intramolecular disulfide bond.

Mouse selenoprotein W (SELENOW) is a small protein containing a selenocysteine (Sec, U) and four cysteine (Cys, C) residues. The Sec residue in SELENOW is located within the conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin (Trx). It is known that glutathione (GSH) binds to SELENOW and that this binding is involved in protecting cells from oxidative stress. However, the regulatory mechanisms controlling the glutathionylation of SELENOW in oxidative stress are unclear. In this study, using purified recombinant SELENOW in which Sec13 was changed to Cys, we found that SELENOW was glutathionylated at Cys33 and that this S-glutathionylation was enhanced by oxidative stress. We also found that the S-glutathionylation of SELENOW at Cys33 in HEK293 cells was due to glutathione S-transferase Pi (GSTpi) and that this modification was reversed by glutaredoxin1 (Grx1). In addition to the disulfide bond between the Cys10 and Cys13 of SELENOW, a second disulfide bond was formed between Cys33 and Cys87 under oxidative stress conditions. The second disulfide bond was reduced by Trx1, but the disulfide bond between Cys10 and Cys13 was not. The second disulfide bond was also reduced by glutathione, but the disulfide bond in the CXXC motif was not. The second disulfide bond of the mutant SELENOW, in which Cys37 was replaced with Ser, was formed at a much lower concentration of hydrogen peroxide than the wild type. We also observed that Cys37 was required for S-glutathionylation, and that S-glutathionylated SELENOW containing Cys37 protected the cells from oxidative stress. Furthermore, the SELENOW (C33, 87S) mutant, which could not form the second disulfide bond, also showed antioxidant activity. Taken together, these results indicate that GSTpi-mediated S-glutathionylation of mouse SELENOW at Cys33 is required for the protection of cells in conditions of oxidative stress, through inhibition of the formation of the second disulfide bond.

PIWI-interacting RNA-36712 restrains breast cancer progression and chemoresistance by interaction with SEPW1 pseudogene SEPW1P RNA.

BACKGROUND: Breast cancer is one of the most common malignancies and the major cause of cancer-related death in women. Although the importance of PIWI-interacting RNAs (piRNAs) in cancer has been increasingly recognized, few studies have been explored the functional mechanism of piRNAs in breast cancer development and progression. METHODS: We examined the top 20 highly expressed piRNAs based on the analysis of TCGA breast cancer data in two patient cohorts to test the roles of piRNAs in breast cancer. The effects of piRNA-36,712 on the malignant phenotypes and chemosensitivity of breast cancer cells were detected in vitro and in vivo. MS2-RIP and reporter gene assays were conducted to identify the interaction and regulation among piRNA-36,712, miRNAs and SEPW1P. Kaplan-Meier estimate with log-rank test was used to compare patient survival by different piRNA-36,712 expression levels. RESULTS: We found piRNA-36,712 level was significantly lower in breast cancer than in normal breast tissues and low level was correlated with poor clinical outcome in patients. Functional studies demonstrated that piRNA-36,712 interacts with RNAs produced by SEPW1P, a retroprocessed pseudogene of SEPW1, and subsequently inhibits SEPW1 expression through competition of SEPW1 mRNA with SEPW1P RNA for microRNA-7 and microRNA-324. We also found that higher SEPW1 expression due to downregulation of piRNA-36,712 in breast cancer may suppress P53, leading to the upregulated Slug but decreased P21 and E-cadherin levels, thus promoting cancer cell proliferation, invasion and migration. Furthermore, we found that piRNA-36,712 had synergistic anticancer effects with the paclitaxel and doxorubicin, two chemotherapeutic agents for breast cancer. CONCLUSIONS: These findings suggest that piRNA-36,712 is a novel tumor suppressor and may serve as a potential predictor for the prognosis of breast cancer patients.

Therapeutic Targeting of the Premetastatic Stage in Human Lung-to-Brain Metastasis.

Brain metastases (BM) result from the spread of primary tumors to the brain and are a leading cause of cancer mortality in adults. Secondary tissue colonization remains the main bottleneck in metastatic development, yet this "premetastatic" stage of the metastatic cascade, when primary tumor cells cross the blood-brain barrier and seed the brain before initiating a secondary tumor, remains poorly characterized. Current studies rely on specimens from fully developed macrometastases to identify therapeutic options in cancer treatment, overlooking the potentially more treatable "premetastatic" phase when colonizing cancer cells could be targeted before they initiate the secondary brain tumor. Here we use our established brain metastasis initiating cell (BMIC) models and gene expression analyses to characterize premetastasis in human lung-to-BM. Premetastatic BMIC engaged invasive and epithelial developmental mechanisms while simultaneously impeding proliferation and apoptosis. We identified the dopamine agonist apomorphine to be a potential premetastasis-targeting drug. In vivo treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes KIF16B, SEPW1, and TESK2 Low expression of these genes was associated with poor survival of patients with lung adenocarcinoma. These results illuminate the cellular and molecular dynamics of premetastasis, which is subclinical and currently impossible to identify or interrogate in human patients with BM. These data present several novel therapeutic targets and associated pathways to prevent BM initiation.Significance: These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases. Cancer Res; 78(17); 5124-34. ©2018 AACR.

Selenomethionine Alleviates AFB1-Induced Damage in Primary Chicken Hepatocytes by Inhibiting CYP450 1A5 Expression via Upregulated SelW Expression.

This study aims to evaluate the protective effects of selenomethionine (SeMet) on aflatoxin B1 (AFB1)-induced hepatotoxicity in primary chicken hepatocytes. Cell viability and lactic dehydrogenase activity assays revealed the dose dependence of AFB1 toxicity to chicken hepatocytes. AFB1 concentrations of >0.05 µg/mL significantly reduced glutathione and total superoxide dismutase levels and increased the malondialdehyde concentration and cytochrome P450 enzyme 1A5 (CYP450 1A5) mRNA levels (P < 0.05). AFB1, however, did not affect CYP450 3A37 mRNA levels. Supplementation with 2 µM SeMet protected against AFB1-induced changes and significantly increased selenoprotein W (SelW) mRNA levels (P < 0.05). Additionally, SelW knockdown attenuated the protective effect of SeMet on AFB1-induced damage and significantly increased the level of CYP450 1A5 expression (P < 0.05). Therefore, SeMet alleviates AFB1-induced damage in primary chicken hepatocytes by improving SelW expression, thus inhibiting CYP450 1A5 expression.

Identification, characterization of selenoprotein W and its mRNA expression patterns in response to somatostatin 14, cysteamine hydrochloride, 17ß-estradiol and a binary mixture of 17ß-estradiol and cysteamine hydrochloride in topmouth culter (Erythroculter ilishaeformis).

In this study, a selenoprotein W cDNA was cloned from topmouth culter (Erythroculter ilishaeformis), and it was designated as EISelW. The EISelW open reading frame was composed of 261 base pairs (bp), encoding 86-amino-acid protein. The 5' untranslated region (UTR) consisted of 104 bp, and the 3'-UTR was composed of 365 bp. A selenocysteine insertion sequence (SECIS) element was found in the 3'-UTR of EISelW mRNA. The SECIS element was classified as form II because of a small additional apical loop presented in SECIS element of EISelW mRNA. Bioinformatic approaches showed that the secondary structure of EISelW was a ß1-α1-ß2-ß3-ß4-α2 pattern from amino-terminal to carboxy-terminal. Real-time PCR analysis of EISelW mRNAs expression in 17 tissues showed that the EISelW mRNA was predominantly expressed in liver, ovary, pituitary, various regions of the brain, spinal cord and head kidney. Study of intraperitoneal injection showed that the levels of EISelW mRNA in brain, liver, ovary and spleen were regulated by somatostatin 14 (SS14), 17ß-estradiol (E2), cysteamine hydrochloride (CSH) and a binary mixture of E2 and CSH, dependent on the dosage. These results suggest that E2, SS14 and CSH status may affect tissues of selenium metabolism by regulating the expression of SelW mRNA, as SelW plays a central role in selenium metabolism.

Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein.

Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein.

Molecular cloning and sequence analysis of selenoprotein W gene and its mRNA expression patterns in response to metabolic status and cadmium exposure in goldfish, Carassius auratus.

Selenoprotein W (SelW) is a low molecular weight and selenocysteine containing protein with redox activity involved in the antioxidant response. In the present study, the full-length cDNA of goldfish (Carassius auratus) selenoprotein W (gfSelW) was successfully cloned from the liver tissue by rapid amplification of cDNA ends technique. The obtained gfSelW cDNA was 730 bp long with a 79 bp 5'-untranslated region (UTR), a 390 bp 3'-UTR containing the consensus polyadenylation signal AATAAA and a 261 bp open reading frame coding a protein of 86 amino acid residues. gfSelW mRNA was observed in all regions of brain and peripheral tissues by semi-quantitative RT-PCR, and the most abundant was detected in testis. After fasting for 1 week, gfSelW mRNA expression levels were significantly decreased compared to the fed group in hypothalamus and liver. After refeeding for 7 days, gfSelW mRNA expression levels were increased back. Furthermore, the mRNA expressions of gfSelW in hypothalamus and liver were varied in periprandial changes and significantly up-regulated after meal 2 h and 4 h, respectively. With cadmium exposure for 24 h, gfSelW mRNA expression levels in gill and leucocytes were significantly decreased at different cadmium concentrations changing from 0.5 ppm to 10 ppm. However, the gfSelW mRNA expression level was sharply increased in liver, relatively to the control about 4.98-fold at 0.5 ppm. The results in this study provide molecular characterization of SelW in goldfish and imply that SelW mRNA expression may be associated with metabolic status and oxidative stress and regulated by metabolic factors and cadmium in fish.

Cloning and expression of selenoprotein W from pearl mussels Cristaria plicata.

Selenoprotein W (SelW) is a selenocysteine containing protein with redox activity involved in the antioxidant response. In this study, a selenoprotein W was cloned from pearl mussel Cristaria plicata (designated as CpSelW), and the expression patterns were characterized in tissues after Aeromonas hydrophila challenged. The full-length cDNA of cpSelW was of 858bp, containing a 5' untranslated region (UTR) of 145bp, a 3' UTR of 455bp with a poly (A) tail, and an open reading frame (ORF) of 258bp encoding a polypeptide of 85 amino acids with the predicted molecular mass of 9.277kDa, which shared 61% identity with SelW from Gallus gallus. A tertiary structure model generated for the CpSelW displayed a ß-α-ß-ß-ß-α secondary structure pattern, which was similar to mouse SelW protein 3D structure. The mRNA of CpSelW was constitutively expressed in tested tissues of healthy mussel, including mantle, gill, hemocytes, muscle, and hepatopancreas, and it was highly expressed in hepatopancreas. After mussels were stimulated by A. hydrophila, the mRNA expression of CpSelW in hemocytes at 6, 12 and 24h, in gill at 12h and in hepatopancreas at 24h was significantly down-regulated.

Selenoprotein W as biomarker for the efficacy of selenium compounds to act as source for selenoprotein biosynthesis.

Selenium is an essential trace element and, like all elements, present in many different compounds with unequivocal functions. This fact is only sporadically mentioned when recommended intake or supplementation is indicated just as "selenium." In mammals, selenium is an integral part of selenoproteins as selenocysteine. Selenocysteine is formed from serine at the respective tRNA((ser)sec), a reaction that requires selenophosphate formed from selenide and ATP. Thus, only compounds that can be metabolized into selenide can serve as sources for selenoprotein biosynthesis. We therefore tested the ability of selenium compounds such as sodium selenite, methylseleninic acid (MeSeA), Se-methyl selenocysteine, and selenomethionine to increase the activity, protein, or mRNA levels of commonly used biomarkers of the selenium status, glutathione peroxidase-1 (GPx1) and thioredoxin reductase, and of putatively new biomarkers, selenoprotein W1 (SepW1), selenoprotein H, and selenoprotein 15 in three different cell lines. Selenite and MeSeA were most efficient in increasing all markers tested, whereas the other compounds had only marginal effects. Effects were higher in the noncancerous young adult mouse colon cells than in the cancer cell lines HepG2 and HT-29. At the protein level, SepW1 responded as well as GPx1 and at the mRNA level, even better. Thus, the outcome of selenium treatment strongly depends on the chemical form, the cell type, and the biomarker used for testing efficacy.

Inhibition of 14-3-3 binding to Rictor of mTORC2 for Akt phosphorylation at Ser473 is regulated by selenoprotein W.

14-3-3 reduces cell proliferation by inhibiting the activity of proteins involved in the signaling pathway that includes Akt kinase. Activation of Akt is enhanced by activating the mammalian target of rapamycin complex 2 (mTORC2). 14-3-3 is also a negative regulator of the mTORC2/Akt pathway, by interacting with a component of mTORC2. Recently, we reported that selenoprotein W (SelW) regulated the interaction between 14-3-3 and its target protein, CDC25B. Here, we show that the binding of Rictor, a component of mTORC2, to 14-3-3, is regulated by the interaction of 14-3-3 with SelW. When SelW was down-regulated, mTORC2-dependent phosphorylation of Akt at Ser473 was decreased. However, the phosphorylation of Thr308 was not affected. The interaction of Rictor with 14-3-3 was increased in SelW-knockdown cells, as compared to control cells. SelW-knockdown cells were also more sensitive to DNA damage induced by etoposide, than control cells. This phenomenon was due to the decreased phosphorylation of Akt at Ser473. We also found that ectopic expression of SelW(U13C) reduced the interaction between Rictor and 14-3-3, leading to Akt phosphorylation at Ser473. Taken together, these findings demonstrate that SelW activates the mTORC2/Akt pathway for Akt phosphorylation at Ser473, by interrupting the binding of Rictor to 14-3-3.

Delayed cell cycle progression in selenoprotein W-depleted cells is regulated by a mitogen-activated protein kinase kinase 4-p38/c-Jun NH2-terminal kinase-p53 pathway.

Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21(Cip1)-dependent G(1)-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G(1) arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G(1) arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G(1) arrest.

Effects of chicken selenoprotein W on H2O2-induced apoptosis in CHO-K1 cells.

Selenoprotein W (SelW) is expressed in various tissues of many animals and acts as an oxidoreductase in mammals. However, little is known about the role of the SelW in birds. To investigate the role of the chicken SelW on H(2)O(2)-induced apoptosis in CHO-K1 cells, overexpression of a chicken SelW cell lines (CHO-K1/SelW) were constructed. Using acridine orange/ethidium bromide (AO/EB) double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays, as well as WST-1 cell viability assay, we monitored the extent of the H(2)O(2)-induced apoptosis and detected the abundance of the caspase-3, caspase-8, and fas mRNA by real-time quantitative reverse transcription PCR (qPCR). We here found that overexpression of SelW cells, compared with the wild-type cells, resulted in a markedly decrease in sensitivity to H(2)O(2)-induced oxidative stress and had a lower apoptotic cell death in AO/EB and TUNEL assays. Cell viability revealed that overexpression of SelW cells had higher cell viability than wild-type cells. qPCR results found that overexpression of SelW cells had a lower levels of caspase-3, caspase-8, and fas mRNA than wild-type cells. Taken together, our findings suggested that SelW could reduce the oxidative damage induced by H(2)O(2) and had an important protective function in against oxidative damage.

Selenoprotein W depletion induces a p53- and p21-dependent delay in cell cycle progression in RWPE-1 prostate epithelial cells.

The anticancer activity of selenium (Se) has been demonstrated in myriad animal and in vitro studies, yet the mechanisms remain obscure. The main form of Se in animal tissues is selenocysteine in selenoproteins, but the relative importance of selenoproteins versus smaller Se compounds in cancer protection is unresolved. Selenoprotein W (SEPW1) is a highly conserved protein ubiquitously expressed in animals, bacteria, and archaea. SEPW1 depletion causes a delay in cell cycle progression at the G1/S transition of the cell cycle in breast and prostate epithelial cells. Tumor suppressor protein p53 is a master regulator of cell cycle progression and is the most frequently mutated gene in human cancers. p53 was increased in SEPW1 silenced cells and was inversely correlated with SEPW1 mRNA in cell lines with altered SEPW1 expression. Silencing SEPW1 decreased ubiquitination of p53 and increased p53 half-life. SEPW1 silencing increased p21(Cip1/WAF1/CDKN1A), while p27 (Kip1/CDKN1B) levels were unaffected. G1-phase arrest from SEPW1 knockdown was abolished by silencing p53 or p21. Cell cycle arrest from SEPW1 silencing was not associated with activation of ATM or phosphorylation of Ser-15 in p53, suggesting the DNA damage response pathway was not involved. Silencing GPX1 had no effect on cell cycle, suggesting that G1-phase arrest from SEPW1 silencing was not due to loss of antioxidant protection. More research is required to identify the function of SEPW1 and how it affects stability of p53.

Delayed cell cycle progression from SEPW1 depletion is p53- and p21-dependent in MCF-7 breast cancer cells.

Selenium (Se) is an essential redox-active trace element with close connections to cancer. Most of Se's biological functions have been attributed to the antioxidant properties of Se-containing proteins. However, the relative contribution of selenoproteins and small Se compounds in cancer protection is still a matter of debate. The tumor suppressor p53 is the most frequently mutated gene in human cancer and is often referred to as the "guardian of the genome". In response to genomic stresses, p53 causes cell cycle arrest to allow time for genomic damage to be repaired before cell division or induces apoptosis to eliminate irreparably damaged cells. Selenoprotein W (SEPW1) is a highly conserved small thioredoxin-like protein required for cell cycle progression. The present work shows that SEPW1 facilitates the G1 to S-phase transition by down-regulating expression of the cyclin-dependent kinase inhibitor p21. SEPW1 controls p21 by modulating levels of the p53 transcription factor, and this is associated with changes in phosphorylation of Ser-33 in p53. More work is needed to identify the mechanism by which SEPW1 regulates phosphorylation of Ser-33 and the kinase or phosphatase enzymes involved.

Characterization and expression of chicken selenoprotein W.

As an essential trace element, selenium (Se) deficiency results in White Muscle Disease in livestock and Keshan disease in humans. The main objectives of this study were to clone and characterize the chicken selenoprotein W (SeW) gene and investigate SeW mRNA expression in chicken tissues. The deduced amino acid (AA) sequence of chicken SeW contains 85 AAs with UAG as the stop codon. Like all SeW genes identified in different species, chicken SeW contains one well-conserved selenocysteine (Sec) at the 13th position encoded by the UGA codon. The proposed glutathione (GSH)-binding site at the Cys(37) of SeW is not conserved in the chicken, but Cys(9) and Sec(13), with possible GSH binding, are conserved in SeWs identified from all species. There are 23-59% and 50-61% homology in cDNA and deduced AA sequences of SeW, respectively, between the chicken and other species. The predicted secondary structure of chicken SeW mRNA indicates that the selenocysteine insertion sequence element is type II with invariant adenosines within the apical bulge. The SeW mRNA expression is high in skeletal muscle followed by brain, but extremely low in other tissues from chickens fed a commercial maize-based diet. The SeW gene is ubiquitously expressed in heart, skeletal muscle, brain, testis, spleen, kidney, lung, liver, stomach and pancreas in chickens fed a commercial diet supplemented with sodium selenite. These results indicate that dietary selenium supplementation regulates SeW gene expression in the chicken and skeletal muscle is the most responsive tissue when dietary Se content is low.

Selenoprotein W depletion in vitro might indicate that its main function is not as an antioxidative enzyme.

Examination of the antioxidative homeostasis in skeletal muscle cells in the presence or absence of selenoprotein W (SelW) is necessary to understand the importance of SelW in the antioxidative system. Depletion of SelW by RNA interference was achieved by introducing a synthetic small interfering RNA into the mouse skeletal muscle cell line C2C12 (C3H). Transfectant screening was performed by real-time reverse transcription-PCR, Western blotting, flow cytometry, fluorescence staining, cell viability, and glutathione assays. SelW expression and mRNA levels were downregulated by 62.1 and 72.4%, respectively. In addition, acute cytotoxicity and an apoptosis rate of approximately 36% in SelW-depleted cells demonstrated that RNA interference was successful. As compared with non-SelW-depleted cells, the enzyme activities of glutathione peroxidase, superoxide dismutase, and catalase and total antioxidative capability and glutathione level increased by 47.6, 103.0, 31.0, 205.6, and 30.0%, respectively (P < 0.05). Thus, SelW is important for the antioxidative system of muscle cells. Depletion of SelW, however, could be compensated by other intracellular antioxidative enzymes because oxidative stress was not the causative factor for apoptosis in SelW-depleted cells. Thus, the main function of SelW in muscle cells is not in the antioxidative system.

Four selenoproteins, protein biosynthesis, and Wnt signalling are particularly sensitive to limited selenium intake in mouse colon.

Selenium is an essential micronutrient. Its recommended daily allowance is not attained by a significant proportion of the population in many countries and its intake has been suggested to affect colorectal carcinogenesis. Therefore, microarrays were used to determine how both selenoprotein and global gene expression patterns in the mouse colon were affected by marginal selenium deficiency comparable to variations in human dietary intakes. Two groups of 12 mice each were fed a selenium-deficient (0.086 mg Se/kg) or a selenium-adequate (0.15 mg Se/kg) diet. After 6 wk, plasma selenium level, liver, and colon glutathione peroxidase (GPx) activity in the deficient group was 12, 34, and 50%, respectively, of that of the adequate group. Differential gene expression was analysed with mouse 44K whole genome microarrays. Pathway analysis by GenMAPP identified the protein biosynthesis pathway as most significantly affected, followed by inflammation, Delta-Notch and Wnt pathways. Selected gene expression changes were confirmed by quantitative real-time PCR. GPx1 and the selenoproteins W, H, and M, responded significantly to selenium intake making them candidates as biomarkers for selenium status. Thus, feeding a marginal selenium-deficient diet resulted in distinct changes in global gene expression in the mouse colon. Modulation of cancer-related pathways may contribute to the higher susceptibility to colon carcinogenesis in low selenium status.