RESUMO
Aging and mechanical overload are prominent risk factors for osteoarthritis (OA), which lead to an imbalance in redox homeostasis. The resulting state of oxidative stress drives the pathological transition of chondrocytes during OA development. However, the specific molecular pathways involved in disrupting chondrocyte redox homeostasis remain unclear. Here, we show that selenophosphate synthetase 1 (SEPHS1) expression is downregulated in human and mouse OA cartilage. SEPHS1 downregulation impairs the cellular capacity to synthesize a class of selenoproteins with oxidoreductase functions in chondrocytes, thereby elevating the level of reactive oxygen species (ROS) and facilitating chondrocyte senescence. Cartilage-specific Sephs1 knockout in adult mice causes aging-associated OA, and augments post-traumatic OA, which is rescued by supplementation of N-acetylcysteine (NAC). Selenium-deficient feeding and Sephs1 knockout have synergistic effects in exacerbating OA pathogenesis in mice. Therefore, we propose that SEPHS1 is an essential regulator of selenium metabolism and redox homeostasis, and its dysregulation governs the progression of OA.
Assuntos
Homeostase , Osteoartrite/genética , Osteoartrite/metabolismo , Fosfotransferases/deficiência , Fosfotransferases/genética , Envelhecimento , Animais , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Knockout , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio , Selênio/metabolismo , Selenoproteínas , TranscriptomaRESUMO
The essential micronutrient Selenium (Se) is co-translationally incorporated as selenocysteine into proteins. Selenoproteins contain one or more selenocysteines and are vital for optimum immunity. Interestingly, many pathogenic bacteria utilize Se for various biological processes suggesting that Se may play a role in bacterial pathogenesis. A previous study had speculated that Francisella tularensis, a facultative intracellular bacterium and the causative agent of tularemia, sequesters Se by upregulating Se-metabolism genes in type II alveolar epithelial cells. Therefore, we investigated the contribution of host vs. pathogen-associated selenoproteins in bacterial disease using F. tularensis as a model organism. We found that F. tularensis was devoid of any Se utilization traits, neither incorporated elemental Se, nor exhibited Se-dependent growth. However, 100% of Se-deficient mice (0.01 ppm Se), which express low levels of selenoproteins, succumbed to F. tularensis-live vaccine strain pulmonary challenge, whereas 50% of mice on Se-supplemented (0.4 ppm Se) and 25% of mice on Se-adequate (0.1 ppm Se) diet succumbed to infection. Median survival time for Se-deficient mice was 8 days post-infection while Se-supplemented and -adequate mice was 11.5 and >14 days post-infection, respectively. Se-deficient macrophages permitted significantly higher intracellular bacterial replication than Se-supplemented macrophages ex vivo, corroborating in vivo observations. Since Francisella replicates in alveolar macrophages during the acute phase of pneumonic infection, we hypothesized that macrophage-specific host selenoproteins may restrict replication and systemic spread of bacteria. F. tularensis infection led to an increased expression of several macrophage selenoproteins, suggesting their key role in limiting bacterial replication. Upon challenge with F. tularensis, mice lacking selenoproteins in macrophages (TrspM) displayed lower survival and increased bacterial burden in the lung and systemic tissues in comparison to WT littermate controls. Furthermore, macrophages from TrspM mice were unable to restrict bacterial replication ex vivo in comparison to macrophages from littermate controls. We herein describe a novel function of host macrophage-specific selenoproteins in restriction of intracellular bacterial replication. These data suggest that host selenoproteins may be considered as novel targets for modulating immune response to control a bacterial infection.
Assuntos
Francisella tularensis/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Selenoproteínas/metabolismo , Tularemia/etiologia , Tularemia/metabolismo , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Francisella tularensis/genética , Francisella tularensis/patogenicidade , Camundongos , Pneumonia/imunologia , Pneumonia/metabolismo , Pneumonia/microbiologia , Pneumonia/patologia , Tularemia/mortalidade , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Trace element selenium (Se) is incorporated as the 21st amino acid, selenocysteine, into selenoproteins through tRNA[Ser]Sec. Selenoproteins act as gatekeepers of redox homeostasis and modulate immune function to effect anti-inflammation and resolution. However, mechanistic underpinnings involving metabolic reprogramming during inflammation and resolution remain poorly understood. Bacterial endotoxin lipopolysaccharide (LPS) activation of murine bone marrow-derived macrophages cultured in the presence or absence of Se (as selenite) was used to examine temporal changes in the proteome and metabolome by multiplexed tandem mass tag-quantitative proteomics, metabolomics, and machine-learning approaches. Kinetic deltagram and clustering analysis indicated that addition of Se led to extensive reprogramming of cellular metabolism upon stimulation with LPS enhancing the pentose phosphate pathway, tricarboxylic acid cycle, and oxidative phosphorylation, to aid in the phenotypic transition toward alternatively activated macrophages, synonymous with resolution of inflammation. Remodeling of metabolic pathways and consequent metabolic adaptation toward proresolving phenotypes began with Se treatment at 0 h and became most prominent around 8 h after LPS stimulation that included succinate dehydrogenase complex, pyruvate kinase, and sedoheptulokinase. Se-dependent modulation of these pathways predisposed bone marrow-derived macrophages to preferentially increase oxidative phosphorylation to efficiently regulate inflammation and its timely resolution. The use of macrophages lacking selenoproteins indicated that all three metabolic nodes were sensitive to selenoproteome expression. Furthermore, inhibition of succinate dehydrogenase complex with dimethylmalonate affected the proresolving effects of Se by increasing the resolution interval in a murine peritonitis model. In summary, our studies provide novel insights into the role of cellular Se via metabolic reprograming to facilitate anti-inflammation and proresolution.
Assuntos
Selênio/metabolismo , Selenoproteínas/metabolismo , Animais , Suscetibilidade a Doenças/metabolismo , Inflamação/metabolismo , Inflamação/fisiopatologia , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peritonite/tratamento farmacológico , Peritonite/imunologia , Proteoma/metabolismo , Proteômica , Selênio/farmacologia , Selenoproteínas/genética , Selenoproteínas/fisiologia , Succinato Desidrogenase/metabolismoRESUMO
Enteropathogenic Escherichia coli (EPEC) leads to adverse colonic inflammation associated with poor resolution of inflammation and loss of epithelial integrity. Micronutrient trace element selenium (Se) is incorporated into selenoproteins as the 21st amino acid, selenocysteine (Sec). Previous studies have shown that such an incorporation of Sec into the selenoproteome is key for the anti-inflammatory functions of Se in macrophages and other immune cells. An intriguing mechanism underlying the anti-inflammatory and pro-resolving effects of Se stems from the ability of selenoproteins to skew arachidonic acid metabolism from pro-inflammatory mediators, prostaglandin E2 (PGE2) toward anti-inflammatory mediators derived from PGD2, such as 15-deoxy-Δ12, 14- prostaglandin J2 (15d-PGJ2), via eicosanoid class switching of bioactive lipids. The impact of Se and such an eicosanoid-class switching mechanism was tested in an enteric infection model of gut inflammation by C. rodentium, a murine equivalent of EPEC. C57BL/6 mice deficient in Se (Se-D) experienced higher mortality when compared to those on Se adequate (0.08 ppm Se) and Se supplemented (0.4 ppm Se) diets following infection. Decreased survival was associated with decreased group 3 innate lymphoid cells (ILC3s) and T helper 17 (Th17) cells in colonic lamina propria of Se-D mice along with deceased expression of epithelial barrier protein Zo-1. Inhibition of metabolic inactivation of PGE2 by 15-prostaglandin dehydrogenase blocked the Se-dependent increase in ILC3 and Th17 cells in addition to reducing epithelial barrier integrity, as seen by increased systemic levels of FITC-dextran following oral administration; while 15d-PGJ2 administration in Se-D mice alleviated the effects by increasing ILC3 and Th17 cells. Mice lacking selenoproteins in monocyte/macrophages via the conditional deletion of the tRNA[Sec] showed increased mortality post infection. Our studies indicate a crucial role for dietary Se in the protection against inflammation following enteric infection via immune mechanisms involving epithelial barrier integrity.
RESUMO
Selenium (Se) is incorporated as the 21st amino acid selenocysteine (Sec) into the growing polypeptide chain of proteins involved in redox gatekeeper functions. Erythropoiesis presents a particular problem to redox regulation as the presence of iron, heme, and unpaired globin chains lead to high levels of free radical-mediated oxidative stress, which are detrimental to erythroid development and can lead to anemia. Under homeostatic conditions, bone marrow erythropoiesis produces sufficient erythrocytes to maintain homeostasis. In contrast, anemic stress induces an alternative pathway, stress erythropoiesis, which rapidly produces new erythrocytes at extramedullary sites, such as spleen, to alleviate anemia. Previous studies suggest that dietary Se protects erythrocytes from such oxidative damage and the absence of selenoproteins causes hemolysis of erythrocytes due to oxidative stress. Furthermore, Se deficiency or lack of selenoproteins severely impairs stress erythropoiesis exacerbating the anemia in rodent models and human patients. Interestingly, erythroid progenitors develop in close proximity with macrophages in structures referred to as erythroblastic islands (EBIs), where macrophage expression of selenoproteins appears to be critical for the expression of heme transporters to facilitate export of heme from macrophage stores to the developing erythroid cells. Here we review the role of Se and selenoproteins in the intrinsic development of erythroid cells in addition to their role in the development of the erythropoietic niche that supports the functional role of EBIs in erythroid expansion and maturation in the spleen during recovery from anemia.
Assuntos
Eritropoese/fisiologia , Selênio/metabolismo , Selenoproteínas/metabolismo , Anemia/metabolismo , Animais , Diferenciação Celular/fisiologia , Eritroblastos/metabolismo , Humanos , Macrófagos/metabolismo , Oxirredução , Estresse Oxidativo/fisiologiaRESUMO
Micronutrient selenium (Se) plays a key role in redox regulation through its incorporation into selenoproteins as the 21st amino acid selenocysteine (Sec). Because Se deficiency appears to be a cofactor in the anemia associated with chronic inflammatory diseases, we reasoned that selenoproteins may contribute to erythropoietic recovery from anemia, referred to as stress erythropoiesis. Here, we report that loss of selenoproteins through Se deficiency or by mutation of the Sec tRNA (tRNA[Sec]) gene (Trsp) severely impairs stress erythropoiesis at 2 stages. Early stress erythroid progenitors failed to expand and properly differentiate into burst-forming unit-erythroid cells , whereas late-stage erythroid progenitors exhibited a maturation defect that affected the transition of proerythroblasts to basophilic erythroblasts. These defects were, in part, a result of the loss of selenoprotein W (SelenoW), whose expression was reduced at both transcript and protein levels in Se-deficient erythroblasts. Mutation of SelenoW in the bone marrow cells significantly decreased the expansion of stress burst-forming unit-erythroid cell colonies, which recapitulated the phenotypes induced by Se deficiency or mutation of Trsp Similarly, mutation of SelenoW in murine erythroblast (G1E) cell line led to defects in terminal differentiation. In addition to the erythroid defects, the spleens of Se-deficient mice contained fewer red pulp macrophages and exhibited impaired development of erythroblastic island macrophages, which make up the niche supporting erythroblast development. Taken together, these data reveal a critical role of selenoproteins in the expansion and development of stress erythroid progenitors, as well as the erythroid niche during acute anemia recovery.
Assuntos
Anemia/metabolismo , Células Precursoras Eritroides/citologia , Eritropoese , Selênio/deficiência , Selenoproteínas/metabolismo , Anemia/genética , Animais , Regulação para Baixo , Eritroblastos/citologia , Eritroblastos/metabolismo , Células Precursoras Eritroides/metabolismo , Camundongos Endogâmicos C57BL , Mutação , Selênio/metabolismo , Selenoproteína W/genética , Selenoproteína W/metabolismo , Selenoproteínas/genética , Baço/citologia , Baço/metabolismoRESUMO
The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trsp(fl/fl)Cre(WT) mice that express selenoproteins driven by tRNA(Sec) (Trsp), whereas N. brasiliensis-infected Trsp(fl/fl)Cre(LysM) selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ(12)-PGJ2 and 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses.
Assuntos
Regulação da Expressão Gênica/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Nippostrongylus/imunologia , Selenoproteínas/imunologia , Infecções por Strongylida/imunologia , Animais , Suplementos Nutricionais , Macrófagos/parasitologia , Macrófagos/patologia , Masculino , Camundongos , Prostaglandina D2/análogos & derivados , Prostaglandina D2/imunologia , Selênio/farmacologia , Infecções por Strongylida/tratamento farmacológicoRESUMO
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.
Assuntos
Interferon gama/sangue , Interleucina-6/sangue , Selênio/administração & dosagem , Selenoproteínas/metabolismo , Animais , Biologia Computacional , Suplementos Nutricionais , Expressão Gênica , Perfilação da Expressão Gênica , Inflamação/imunologia , Interferon gama/imunologia , Interleucina-6/imunologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Selênio/sangue , Selenoproteínas/genética , Análise de Sequência de RNA , Selenito de Sódio/metabolismo , Regulação para CimaRESUMO
Acetylation of histone and non-histone proteins by histone acetyltransferases plays a pivotal role in the expression of proinflammatory genes. Given the importance of dietary selenium in mitigating inflammation, we hypothesized that selenium supplementation may regulate inflammatory gene expression at the epigenetic level. The effect of selenium towards histone acetylation was examined in both in vitro and in vivo models of inflammation by chromatin immunoprecipitation assays and immunoblotting. Our results indicated that selenium supplementation, as selenite, decreased acetylation of histone H4 at K12 and K16 in COX-2 and TNFα promoters, and of the p65 subunit of the redox sensitive transcription factor NFκB in primary and immortalized macrophages. On the other hand, selenomethionine had a much weaker effect. Selenite treatment of HIV-1-infected human monocytes also significantly decreased the acetylation of H4 at K12 and K16 on the HIV-1 promoter, supporting the down-regulation of proviral expression by selenium. A similar decrease in histone acetylation was also seen in the colonic extracts of mice treated with dextran sodium sulfate that correlated well with the levels of selenium in the diet. Bone-marrow-derived macrophages from Trsp(fl/fl)Cre(LysM) mice that lack expression of selenoproteins in macrophages confirmed the important role of selenoproteins in the inhibition of histone H4 acetylation. Our studies suggest that the ability of selenoproteins to skew the metabolism of arachidonic acid contributes, in part, to their ability to inhibit histone acetylation. In summary, our studies suggest a new role for selenoproteins in the epigenetic modulation of proinflammatory genes.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Histonas/metabolismo , Macrófagos/metabolismo , Regiões Promotoras Genéticas , Selênio/metabolismo , Selenoproteínas/metabolismo , Acetilação , Animais , Linhagem Celular Transformada , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Suplementos Nutricionais , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Selênio/deficiência , Selênio/uso terapêutico , Selenometionina/administração & dosagem , Selenoproteínas/genética , Selenito de Sódio/administração & dosagem , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress proinflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% dextran sodium sulfate in wild-type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.08 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Selenocysteinyl transfer RNA knockout mice (Trsp(fl/fl)LysM(Cre)) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of proinflammatory and anti-inflammatory genes, and modulation of PG metabolites in urine and plasma. Whereas Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an upregulation of expression of proinflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and proresolving effects. Selenoproteins in macrophages protect mice from dextran sodium sulfate-colitis by enhancing 15-PGDH-dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.
Assuntos
Colite/imunologia , Macrófagos/imunologia , Selenoproteínas/imunologia , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/toxicidade , Suplementos Nutricionais , Dinoprostona/genética , Dinoprostona/imunologia , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/imunologia , Inflamação/genética , Inflamação/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/imunologia , Selênio/farmacologia , Selenoproteínas/genéticaRESUMO
S-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA(Sec) to the Um34 form. The formation of methylated tRNA(Sec) facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA(Sec) and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA(Sec) hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.
Assuntos
Células Endoteliais/enzimologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Metiltransferases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , S-Adenosil-Homocisteína/metabolismo , Adesão Celular/fisiologia , Células Endoteliais/efeitos dos fármacos , Homocisteína/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peróxido de Hidrogênio/metabolismo , Leucócitos/citologia , Metilação , Estresse Oxidativo/fisiologia , RNA de Transferência de Serina/metabolismo , S-Adenosilmetionina/metabolismo , Selênio/farmacologia , Selenoproteínas/metabolismo , Glutationa Peroxidase GPX1RESUMO
The many biological and biomedical effects of selenium are relatively unknown outside the selenium field. This fascinating element, initially described as a toxin, was subsequently shown to be essential for health and development. By the mid-1990s selenium emerged as one of the most promising cancer chemopreventive agents, but subsequent human clinical trials yielded contradictory results. However, basic research on selenium continued to move at a rapid pace, elucidating its many roles in health, development, and in cancer prevention and promotion. Dietary selenium acts principally through selenoproteins, most of which are oxidoreductases involved in diverse cellular functions.
Assuntos
Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Selênio/metabolismo , Selenocisteína/metabolismo , Selenoproteínas/metabolismo , HumanosRESUMO
Incorporation of selenium into ~25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA([Ser]Sec) Um34 methylation. Furthermore, we find evidence for translation in the 5'-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.
Assuntos
Códon de Terminação/metabolismo , Suplementos Nutricionais , Biossíntese de Proteínas/fisiologia , Selênio/farmacologia , Selenocisteína/metabolismo , Selenoproteínas/biossíntese , Animais , Códon de Terminação/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência Aminoácido-Específico/metabolismo , Selenocisteína/genéticaRESUMO
Changes in dietary selenium and selenoprotein status may influence both anti- and pro-cancer pathways, making the outcome of interventions different from one study to another. To characterize such outcomes in a defined setting, we undertook a controlled hepatocarcinogenesis study involving varying levels of dietary selenium and altered selenoprotein status using mice carrying a mutant (A37G) selenocysteine tRNA transgene (Trsp(tG37) ) and/or a cancer driver TGFα transgene. The use of Trsp(tG37) altered selenoprotein expression in a selenoprotein and tissue specific manner and, at sufficient dietary selenium levels, separate the effect of diet and selenoprotein status. Mice were maintained on diets deficient in selenium (0.02 ppm selenium) or supplemented with 0.1, 0.4 or 2.25 ppm selenium or 30 ppm triphenylselenonium chloride (TPSC), a non-metabolized selenium compound. Trsp(tG37) transgenic and TGFα/Trsp(tG37) bi-transgenic mice subjected to selenium-deficient or TPSC diets developed a neurological phenotype associated with early morbidity and mortality prior to hepatocarcinoma development. Pathology analyses revealed widespread disseminated pyogranulomatous inflammation. Pyogranulomas occurred in liver, lungs, heart, spleen, small and large intestine, and mesenteric lymph nodes in these transgenic and bi-transgenic mice. The incidence of liver tumors was significantly increased in mice carrying the TGFα transgene, while dietary selenium and selenoprotein status did not affect tumor number and multiplicity. However, adenoma and carcinoma size and area were smaller in TGFα transgenic mice that were fed 0.4 and 2.25 versus 0.1 ppm of selenium. Thus, selenium and selenoprotein deficiencies led to widespread pyogranuloma formation, while high selenium levels inhibited the size of TGFα-induced liver tumors.
Assuntos
Suplementos Nutricionais , Granuloma/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Selênio/deficiência , Selênio/uso terapêutico , Selenoproteínas/deficiência , Animais , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Granuloma/sangue , Isótopos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/sangue , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , RNA de Transferência Aminoácido-Específico/metabolismo , Selênio/sangue , Selenoproteínas/metabolismo , Fator de Crescimento Transformador alfaRESUMO
Selenium (Se) has long been known for its cancer prevention properties, but the molecular basis remains unclear. The principal questions in assessing the effect of dietary Se in cancer are whether selenoproteins, small molecule selenocompounds, or both, are involved, and under which conditions and genotypes Se may be protective. In this study, we examined diethylnitrosamine-induced hepatocarcinogenesis in mice lacking a subset of selenoproteins due to expression of a mutant selenocysteine tRNA gene (Trsp (A37G) mice). To uncouple the effects of selenocompounds and selenoproteins, these animals were examined at several levels of dietary Se. Our analysis revealed that tumorigenesis in Trsp (A37G) mice maintained on the adequate Se diet was increased. However, in the control, wild-type mice, both Se deficiency and high Se levels protected against tumorigenesis. We further found that the Se-deficient diet induced severe neurological phenotypes in Trsp A37G mice. Surprisingly, a similar phenotype could be induced in these mice at high dietary Se intake. Overall, our results show a complex role of Se in chemically induced hepatocarcinogenesis, which involves interaction among selenoproteins, selenocompounds and toxins, and depends on genotype and background of the animals.
Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/prevenção & controle , Selênio/administração & dosagem , Selenoproteínas/genética , Selenoproteínas/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Dieta , Feminino , Genótipo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , RNA de Transferência Aminoácido-Específico/genéticaRESUMO
AIM: Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. RESULTS: Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA([Ser]Sec) and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. INNOVATION: We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. CONCLUSION: XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution.
Assuntos
Glutationa Peroxidase/genética , Túbulos Renais Proximais/metabolismo , Selênio/metabolismo , Animais , Membrana Basal/enzimologia , Membrana Basal/metabolismo , Microanálise por Sonda Eletrônica , Técnicas de Inativação de Genes , Glutationa Peroxidase/metabolismo , Rim/metabolismo , Túbulos Renais Proximais/enzimologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos-Toupeira , Aminoacil-RNA de Transferência/genética , Selenoproteína P/genética , Espectrometria por Raios XRESUMO
Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA([Ser]Sec) and its insertion was dependent on the Sec insertion sequence element in the 3'UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA([Ser]Sec) to generate Cys-tRNA([Ser]Sec). Cys was inserted in vivo at UGA codons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinery-based mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.
Assuntos
Códon de Terminação , Cisteína/biossíntese , Cisteína/genética , Selenocisteína/metabolismo , Animais , Dieta , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Células NIH 3T3 , Fosfatos/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Selênio/administração & dosagem , Selênio/metabolismo , Selenocisteína/genética , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.
Assuntos
Queratinócitos/citologia , Queratinócitos/metabolismo , Selenoproteínas/metabolismo , Pele/crescimento & desenvolvimento , Pele/metabolismo , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Adesão Celular , Diferenciação Celular , Proliferação de Células , Células Epidérmicas , Epiderme/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Queratina-14/genética , Queratina-14/metabolismo , Peroxidação de Lipídeos , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Camundongos , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência Aminoácido-Específico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética , Selênio/deficiência , Selenoproteínas/deficiência , Selenoproteínas/genética , Pele/citologiaRESUMO
Selenium (Se) has been known for many years to have played a role in boosting the immune function, but the manner in which this element acts at the molecular level in host defence and inflammatory diseases is poorly understood. To elucidate the role of Se-containing proteins in the immune function, we knocked out the expression of this protein class in T-cells or macrophages of mice by targeting the removal of the selenocysteine tRNA gene using loxP-Cre technology. Mice with selenoprotein-less T-cells manifested reduced pools of mature and functional T-cells in lymphoid tissues and an impairment in T-cell-dependent antibody responses. Furthermore, selenoprotein deficiency in T-cells led to an inability of these cells to suppress reactive oxygen species production, which in turn affected their ability to proliferate in response to T-cell receptor stimulation. Selenoprotein-less macrophages, on the other hand, manifested mostly normal inflammatory responses, but this deficiency resulted in an altered regulation in extracellular matrix-related gene expression and a diminished migration of macrophages in a protein gel matrix. These observations provided novel insights into the role of selenoproteins in the immune function and tissue homeostasis.
Assuntos
Imunidade/fisiologia , Macrófagos/metabolismo , Selênio/imunologia , Selenoproteínas/imunologia , Linfócitos T/metabolismo , Animais , Anticorpos/sangue , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , RNA de Transferência , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Selênio/metabolismo , Selenocisteína/genética , Selenoproteínas/genética , Selenoproteínas/metabolismoRESUMO
Cerebral selenium (Se) deficiency is associated with neurological phenotypes including seizures and ataxia. We wanted to define whether neurons require selenoprotein expression and which selenoproteins are most important, and explore the possible pathomechanism. Therefore, we abrogated the expression of all selenoproteins in neurons by genetic inactivation of the tRNA[Ser](Sec) gene. Cerebral expression of selenoproteins was significantly diminished in the mutants, and histological analysis revealed progressive neurodegeneration. Developing interneurons failed to specifically express parvalbumin (PV) in the mutants. Electrophysiological recordings, before overt cell death, showed normal excitatory transmission, but revealed spontaneous epileptiform activity consistent with seizures in the mutants. In developing cortical neuron cultures, the number of PV(+) neurons was reduced on combined Se and vitamin E deprivation, while other markers, such as calretinin (CR) and GAD67, remained unaffected. Because of the synergism between Se and vitamin E, we analyzed mice lacking neuronal expression of the Se-dependent enzyme glutathione peroxidase 4 (GPx4). Although the number of CR(+) interneurons remained normal in Gpx4-mutant mice, the number of PV(+) interneurons was reduced. Since these mice similarly exhibit seizures and ataxia, we conclude that GPx4 is a selenoenzyme modulating interneuron function and PV expression. Cerebral SE deficiency may thus act via reduced GPx4 expression.-Wirth, E. K., Conrad, M., Winterer, J., Wozny, C., Carlson, B. A., Roth, S., Schmitz, D., Bornkamm, G. W., Coppola, V., Tessarollo, L., Schomburg, L., Köhrle, J., Hatfield, D. L., Schweizer, U. Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration.