RESUMO
Selenoprotein I (SELENOI) catalyzes the final reaction of the CDP-ethanolamine branch of the Kennedy pathway, generating the phospholipids phosphatidylethanolamine (PE) and plasmenyl-PE. Plasmenyl-PE is a key component of myelin and is characterized by a vinyl ether bond that preferentially reacts with oxidants, thus serves as a sacrificial antioxidant. In humans, multiple loss-of-function mutations in genes affecting plasmenyl-PE metabolism have been implicated in hereditary spastic paraplegia, including SELENOI. Herein, we developed a mouse model of nervous system-restricted SELENOI deficiency that circumvents embryonic lethality caused by constitutive deletion and recapitulates phenotypic features of hereditary spastic paraplegia. Resulting mice exhibited pronounced alterations in brain lipid composition, which coincided with motor deficits and neuropathology including hypomyelination, elevated reactive gliosis, and microcephaly. Further studies revealed increased lipid peroxidation in oligodendrocyte lineage cells and disrupted oligodendrocyte maturation both in vivo and in vitro. Altogether, these findings detail a critical role for SELENOI-derived plasmenyl-PE in myelination that is of paramount importance for neurodevelopment.
Assuntos
Homeostase , Metabolismo dos Lipídeos , Bainha de Mielina , Oligodendroglia , Selenoproteínas , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Encéfalo/patologia , Peroxidação de Lipídeos , Camundongos Knockout , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Fosfatidiletanolaminas/metabolismo , Éteres Fosfolipídicos/metabolismo , Plasmalogênios/metabolismo , Selenoproteínas/metabolismo , Selenoproteínas/genética , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologiaRESUMO
While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.
Assuntos
Sistema de Sinalização das MAP Quinases , MicroRNAs , Linfócitos T , Regiões 3' não Traduzidas , Animais , Ativação Linfocitária , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/imunologia , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , MicroRNAs/genética , MicroRNAs/imunologia , MicroRNAs/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Elk-1 do Domínio ets/imunologia , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Redox control of protein function involves oxidation and reduction of amino acid residues, but the mechanisms and regulators involved are insufficiently understood. Here, we report that in conjunction with Mical proteins, methionine-R-sulfoxide reductase B1 (MsrB1) regulates mammalian actin assembly via stereoselective methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin are specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by selenoprotein MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilize this redox control during cellular activation by stimulating MsrB1 expression and activity as a part of innate immunity. We identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study shows that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation.
Assuntos
Actinas/metabolismo , Macrófagos/metabolismo , Metionina Sulfóxido Redutases/genética , Metionina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Oxigenases de Função Mista/metabolismo , Oxirredutases/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos , Oxirredução , Estresse Oxidativo , Oxirredutases/genéticaRESUMO
Selenoprotein I (SELENOI) is an ethanolamine phosphotransferase that catalyzes the third reaction of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Since the role of SELENOI in murine embryogenesis has not been investigated, SELENOI-/+ mating pairs were used to generate global KO offspring. Of 323 weanling pups, no homozygous KO genotypes were found. E6.5-E18.5 embryos (165 total) were genotyped, and only two E18.5 KO embryos were detected with no discernable anatomical defects. To screen embryos prior to uterine implantation that occurs ~ E6, blastocyst embryos (E3.5-E4.4) were flushed from uteruses of pregnant females and analyzed for morphology and genotype. KO embryos were detected in 5 of 6 pregnant females, and 7 of the 32 genotyped embryos were found to be SELENOI KO that exhibited no overt pathological features. Overall, these results demonstrate that, except for rare cases (2/490 = 0.4%), global SELENOI deletion leads to early embryonic lethality.
Assuntos
Blastocisto/patologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos/embriologia , Animais , Animais Recém-Nascidos , Blastocisto/ultraestrutura , Implantação do Embrião , Perda do Embrião/genética , Perda do Embrião/patologia , Desenvolvimento Embrionário , Etanolaminofosfotransferase , Feminino , Deleção de Genes , Homozigoto , Masculino , Camundongos/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , GravidezRESUMO
Calcium (Ca(2+)) is a secondary messenger in cells and Ca(2+) flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca(2+) flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca(2+) flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially palmitoylated cysteine, and mutation of these three cysteines to alanines resulted in decreased IP3R palmitoylation and function. These findings reveal IP3R palmitoylation as a critical regulator of Ca(2+) flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.
Assuntos
Aciltransferases/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Processamento de Proteína Pós-Traducional , Selenoproteínas/fisiologia , Subpopulações de Linfócitos T/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Animais , Células da Medula Óssea/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Cisteína/química , Retículo Endoplasmático/enzimologia , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Células Jurkat , Lipoilação , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/farmacologia , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Selênio/fisiologia , Selenoproteínas/química , Selenoproteínas/deficiência , Tapsigargina/farmacologia , Transfecção , Domínios de Homologia de srcRESUMO
To study the effect of the micronutrient selenium on malignant mesothelioma (MM) progression, we cultured four different MM cell lines in media containing increasing amounts of sodium selenite (30, 50, and 80 nmol/L). Increasing selenium levels increased density-dependent proliferation and mobility for CRH5 and EKKH5 but not AB12 and AK7. Comparing these cell lines revealed that extracellular regulated kinase (ERK) phosphorylation was sensitive to a selenium increase in CRH5 and EKKH5 but not AB12 and AK7 cells. Stable expression of a dominant-negative mutant ERK eliminated the effects of increasing selenium. Because ERK is redox sensitive, we compared the MM cell lines in terms of glutathione levels and the capacity to reduce exogenous hydrogen peroxide. Increasing selenium levels led to higher glutathione and reducing capacity in CRH5 and EKKH5 but not AB12 and AK7. The reducing agent N-acetylcysteine eliminated the effects of selenium on ERK activation, proliferation, and mobility. Mice fed diets containing increasing levels of selenium (0.08, 0.25, and 1.0 ppm) showed increased tumor progression for CRH5 but not AB12, MM cells, and in vivo N-acetylcysteine treatment eliminated these effects. These data suggest that the effects of dietary selenium on MM tumor progression depend on the arising cancer cells' redox metabolism, and the tumors able to convert increased selenium into a stronger reducing capacity actually benefit from increased selenium intake.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Micronutrientes/metabolismo , Selênio/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Dieta , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática/fisiologia , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxirredução , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated, reconstituted with either CAST-knockout (KO) or wild-type (WT) bone marrow, and subjected to dextran sulfate sodium-induced colitis. CAST-KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared with WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNF-α, and IFN-γ mRNA levels compared with WT controls. Mechanistic investigations using small interfering RNA and KO bone marrow to generate CAST-deficient macrophages showed that CAST deficiency during activation with bacterial pathogen associated molecular patterns, including heat-killed Enterococcus faecalis or CpG DNA, led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNF-α secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.
Assuntos
Proteínas de Ligação ao Cálcio/farmacologia , Colite/imunologia , Colite/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Animais , Colite/genética , Colite/patologia , Inibidores de Cisteína Proteinase/farmacologia , Citocinas/biossíntese , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Transporte Proteico , Transdução de SinaisRESUMO
The mechanisms driving metabolic reprogramming during B cell activation are unclear, particularly roles for enzymatic pathways involved in lipid remodeling. We found that murine B cell activation with lipopolysaccharide (LPS) led to a 1.6-fold increase in total lipids that included higher levels of phosphatidylethanolamine (PE) and plasmenyl PE. Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase involved in the synthesis of both PE and plasmenyl PE, and SELENOI expression was also upregulated during activation. Selenoi knockout (KO) B cells exhibited decreased levels of plasmenyl PE, which plays an important antioxidant role. Lipid peroxidation was measured and found to increase â¼2-fold in KO vs. wild-type (WT) B cells. Cell death was not impacted by KO in LPS-treated B cells and proliferation was only slightly reduced, but differentiation into CD138 + Blimp-1+ plasma B cells was decreased â¼2-fold. This led to examination of B cell receptors important for differentiation that recognize the ligand B cell activating factor, and levels of TACI (transmembrane activator, calcium-modulator, and cytophilin ligand interactor) (CD267) were significantly decreased on KO B cells compared with WT control cells. Vaccination with ovalbumin/adjuvant led to decreased ovalbumin-specific immunoglobulin M (IgM) levels in sera of KO mice compared with WT mice. Real-time polymerase chain reaction analyses revealed a decreased switch from surface to secreted IgM in spleens of KO mice induced by vaccination or LP-BM5 retrovirus infection. Overall, these findings detail the lipidomic response of B cells to LPS activation and reveal the importance of upregulated SELENOI for promoting differentiation into IgM-secreting plasma B cells.
Assuntos
Linfócitos B , Diferenciação Celular , Imunoglobulina M , Lipopolissacarídeos , Ativação Linfocitária , Selenoproteínas , Animais , Lipopolissacarídeos/farmacologia , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Camundongos , Selenoproteínas/metabolismo , Selenoproteínas/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Camundongos Knockout , Plasmócitos/metabolismo , Plasmócitos/imunologia , Lipidômica , Regulação para Cima , Camundongos Endogâmicos C57BLRESUMO
Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Cálcio/metabolismo , Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Fagocitose/fisiologia , Animais , Complexo Antígeno-Anticorpo/farmacologia , Antivirais/farmacologia , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interferon gama/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fagocitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Receptores de IgG/metabolismoRESUMO
Selenoprotein K (Sel K) is a selenium-containing protein for which no function has been identified. We found that Sel K is an endoplasmic reticulum transmembrane protein expressed at relatively high levels in immune cells and is regulated by dietary selenium. Sel K(-/-) mice were generated and found to be similar to wild-type controls regarding growth and fertility. Immune system development was not affected by Sel K deletion, but specific immune cell defects were found in Sel K(-/-) mice. Receptor-mediated Ca(2+) flux was decreased in T cells, neutrophils, and macrophages from Sel K(-/-) mice compared with controls. Ca(2+)-dependent functions including T cell proliferation, T cell and neutrophil migration, and Fcγ receptor-mediated oxidative burst in macrophages were decreased in cells from Sel K(-/-) mice compared with that in cells from controls. West Nile virus infections were performed, and Sel K(-/-) mice exhibited decreased viral clearance in the periphery and increased viral titers in brain. Furthermore, West Nile virus-infected Sel K(-/-) mice demonstrated significantly lower survival (2 of 23; 8.7%) compared with that of wild-type controls (10 of 26; 38.5%). These results establish Sel K as an endoplasmic reticulum-membrane protein important for promoting effective Ca(2+) flux during immune cell activation and provide insight into molecular mechanisms by which dietary selenium enhances immune responses.
Assuntos
Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Cálcio/fisiologia , Inibição de Migração Celular/imunologia , Selenoproteínas/deficiência , Selenoproteínas/genética , Animais , Cálcio/antagonistas & inibidores , Inibição de Migração Celular/genética , Modelos Animais de Doenças , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Regulação da Expressão Gênica/imunologia , Humanos , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Peritonite/genética , Peritonite/imunologia , Peritonite/patologia , Receptores de Peptídeos/metabolismo , Selênio/administração & dosagem , Selênio/fisiologia , Selenoproteínas/biossíntese , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Calpains are proteolytic enzymes that modulate cellular function through cleavage of targets, thereby modifying their actions. An important role is emerging for calpains in regulating inflammation and immune responses, although specific mechanisms by which this occurs have not been clearly defined. In this study, we identify a novel target of calpain, selenoprotein K (SelK), which is an endoplasmic reticulum transmembrane protein important for Ca(2+) flux in immune cells. Calpain-mediated cleavage of SelK was detected in myeloid cells (macrophages, neutrophils, and dendritic cells) but not in lymphoid cells (B and T cells). Both m- and µ-calpain were capable of cleaving immunoprecipitated SelK, but m-calpain was the predominant isoform expressed in mouse immune cells. Consistent with these results, specific inhibitors were used to show that only m-calpain cleaved SelK in macrophages. The cleavage site in SelK was identified between Arg(81) and Gly(82) and the resulting truncated SelK was shown to lack selenocysteine, the amino acid that defines selenoproteins. Resting macrophages predominantly expressed cleaved SelK and, when activated through different Toll-like receptors (TLRs), SelK cleavage was inhibited. We found that decreased calpain cleavage was due to TLR-induced up-regulation of the endogenous inhibitor, calpastatin. TLR-induced calpastatin expression not only inhibited SelK cleavage, but cleavage of another calpain target, talin. Moreover, the expression of the calpain isoforms and calpastatin in macrophages were different from T and B cells. Overall, our findings identify SelK as a novel calpain target and reveal dynamic changes in the calpain/calpastatin system during TLR-induced activation of macrophages.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Macrófagos/metabolismo , Selenoproteínas/metabolismo , Receptores Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Células da Medula Óssea/citologia , Cálcio/metabolismo , Movimento Celular , Retículo Endoplasmático/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase contributing to cellular metabolism and the synthesis of glycosylphosphatidylinositol (GPI) anchors. SELENOI knockout (KO) in T cells has been shown to impair metabolic reprogramming during T cell activation and reduce GPI-anchored Thy-1 levels, which are both crucial for Th17 differentiation. This suggests SELENOI may be important for Th17 differentiation, and we found that SELENOI was indeed up-regulated early during the activation of naïve CD4+ T cells in Th17 conditions. SELENOI KO reduced RORγt mRNA levels by decreasing SOX5 and STAT3 binding to promoter and enhancer regions in the RORC gene encoding this master regulator of Th17 cell differentiation. Differentiation of naïve CD4+ T cells into inflammatory versus tolerogenic Th cell subsets was analyzed and results showed that SELENOI deficiency skewed differentiation away from pathogenic Th17 cells (RORγt+ and IL-17A+ ) while promoting tolerogenic phenotypes (Foxp3+ and IL-10+ ). Wild-type and T cell-specific SELENOI KO mice were subjected to experimental autoimmune encephalitis (EAE), with KO mice exhibiting diminished clinical symptoms, reduced CNS pathology and decreased T cell infiltration. Flow cytometry showed that SELENOI T cell KO mice exhibited lower CD4+ RORγt+ and CD4+ IL-17A+ T cells and higher CD4+ CD25+ FoxP3+ T cells in CNS tissues of mice subjected to EAE. Thus, the metabolic enzyme SELENOI is up-regulated to promote RORγt transcription that drives Th17 differentiation, and SELENOI deficiency shifts differentiation toward tolerogenic phenotypes while protecting against pathogenic Th17 responses.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Células Th17 , Camundongos , Animais , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Interleucina-17/metabolismo , Diferenciação Celular , Camundongos Knockout , Fatores de Transcrição Forkhead/metabolismo , Fenótipo , Selenoproteínas/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Selenium (Se) is thought to confer cardioprotective effects through the actions of antioxidant selenoprotein enzymes that directly limit levels of ROS such as hydrogen peroxide (H(2)O(2)) or that reverse oxidative damage to lipids and proteins. To determine how the selenoproteome responds to myocardial hypertrophy, two mouse models were employed: triidothyronine (T3)- or isoproterenol (ISO)-treatment. After 7days of T3- and ISO-treatment, cardiac stress was demonstrated by increased H(2)O(2) and caspase-3 activity. Neither treatment produced significant increases in phospholipid peroxidation or TUNEL-positive cells, suggesting that antioxidant systems were protecting the cardiomyocytes from damage. Many selenoprotein mRNAs were induced by T3- and ISO-treatment, with levels of methionine sulfoxide reductase 1 (MsrB1, also called SelR) mRNA showing the largest increases. MsrB enzymatic activity was also elevated in both models of cardiac stress, while glutathione peroxidase (GPx) activity and thioredoxin reductase (Trxrd) activity were moderately and nonsignificantly increased, respectively. Western blot assays revealed a marked increase in MsrB1 and moderate increases in GPx3, GPx4, and Trxrd1, particularly in T3-treated hearts. Thus, the main response of the selenoproteome during hypertrophy does not involve increased GPx1, but increased GPx3 for reducing extracellular H(2)O(2) and increased GPx4, Trxrd1, and MsrB1 for minimizing intracellular oxidative damage.
Assuntos
Antioxidantes/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Selenoproteínas/metabolismo , Animais , Hipertrofia/induzido quimicamente , Hipertrofia/metabolismo , Hipertrofia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , RNA Mensageiro/genética , Selenoproteínas/genética , Regulação para CimaRESUMO
OBJECTIVE: T cell activation triggers metabolic reprogramming to meet increased demands for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cells and cancer cells, which led us to investigate its potential role during T cell activation. METHODS: As selenoprotein I (SELENOI) is an enzyme participating in two metabolic pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI-deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation. RESULTS: SELENOI knockout (KO) in mouse T cells led to reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor AMPK. AMPK was inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment CONCLUSIONS: T cell activation upregulates SELENOI-dependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.
Assuntos
Etanolamina/metabolismo , Fosfolipídeos/biossíntese , Selenoproteínas/metabolismo , Linfócitos T/metabolismo , Animais , Proliferação de Células , Etanolaminas/metabolismo , Feminino , Glicólise , Glicosilfosfatidilinositóis/metabolismo , Lipogênese/genética , Lipogênese/fisiologia , Masculino , Redes e Vias Metabólicas , Metabolômica , Camundongos , Camundongos Knockout , Fosfatidiletanolaminas/metabolismo , Selenoproteínas/deficiência , Selenoproteínas/genéticaRESUMO
The immune-enhancing effects of selenium (Se) supplementation make it a promising complementary and alternative medicine modality for boosting immunity, although mechanisms by which Se influences immunity are unclear. Mice fed low (0.08 mg/kg), medium (0.25 mg/kg), or high (1.0 mg/kg) Se diets for 8 wk were challenged with peptide/adjuvant. Antigen-specific CD4(+) T cell responses were increased in the high Se group compared with the low and medium Se groups. T cell receptor signaling in ex vivo CD4(+) T cells increased with increasing dietary Se, with all 3 groups differing from one another in terms of calcium mobilization, oxidative burst, translocation of nuclear factor of activated T cells, and proliferation. The high Se diet increased expression of interleukin (IL)-2 and the high affinity chain of the IL-2 receptor compared with the low and medium Se diets. The high Se diet skewed the T helper (Th)1/Th2 balance toward a Th1 phenotype, leading to higher interferon-gamma and CD40 ligand levels compared with the low and medium Se diets. Prior to CD4(+) T cell activation, levels of reactive oxygen species did not differ among the groups, but the low Se diet decreased free thiols compared with the medium and high Se diets. Addition of exogenous free thiols eliminated differences in CD4(+) T cell activation among the dietary groups. Overall, these data suggest that dietary Se levels modulate free thiol levels and specific signaling events during CD4(+) T cell activation, which influence their proliferation and differentiation.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Suplementos Nutricionais , Ativação Linfocitária , Selênio/administração & dosagem , Selênio/farmacologia , Compostos de Sulfidrila/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica/fisiologia , Interleucina-2/genética , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismoRESUMO
Selenoprotein P (Sel P) is a selenium-rich glycoprotein believed to play a key role in selenium (Se) transport throughout the body. Development of a Sel P knockout mouse model has supported this notion and initial studies have indicated that selenium supply to various tissues is differentially affected by genetic deletion of Sel P. Se in the form of the amino acid, selenocysteine, is incorporated into selenoproteins at UGA codons. Thus, Se availability affects not only selenoprotein levels, but also the turnover of selenoprotein mRNAs via the nonsense-mediated decay pathway. We investigated how genetic deletion of Sel P in mice affected levels of the mRNAs encoding all known members of the murine selenoprotein family, as well as three non-selenoprotein factors involved in their synthesis, selenophosphate synthetase 1 (SPS1), SECIS-binding protein 2 (SBP2) and SECp43. Our findings present a comprehensive description of selenoprotein mRNA expression in the following murine tissues: brain, heart, intestine, kidney, liver, lung, spleen and testes. We also describe how abundance of selenoproteins and selenoprotein-synthesis factors are affected by genetic deletion of Sel P in some of these tissues, providing insight into how the presence of this selenoprotein influences selenoprotein mRNA levels, and thus, the selenoproteome.
Assuntos
Selênio/metabolismo , Selenoproteína P/genética , Selenoproteínas/metabolismo , Animais , Encéfalo/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Proteoma/metabolismo , RNA Mensageiro/metabolismo , Selenoproteína P/fisiologia , Selenoproteínas/genética , Testículo/metabolismo , Distribuição TecidualRESUMO
Galectin-9 has emerged as a promising biological target for cancer immunotherapy due to its role as a regulator of macrophage and T-cell differentiation. In addition, its expression in tumor cells modulates tumor cell adhesion, metastasis, and apoptosis. Malignant mesothelioma (MM) is an aggressive neoplasm of the mesothelial cells lining the pleural and peritoneal cavities, and in this study, we found that both human MM tissues and mouse MM cells express high levels of galectin-9. Using a novel monoclonal antibody (mAb) (Clone P4D2) that binds the C-terminal carbohydrate recognition domain (CRD) of galectin-9, we demonstrate unique agonistic properties resulting in MM cell apoptosis. Furthermore, the P4D2 mAb reduced tumor-associated macrophages differentiation toward a protumor phenotype. Importantly, these effects exerted by the P4D2 mAb were observed in both human and mouse in vitro experiments and not observed with another antigalectin-9 specific mAb (clone P1D9) that engages the N-terminus CRD of galectin-9. In syngeneic murine models of MM, P4D2 mAb treatment inhibited tumor growth and improved survival, with tumors from P4D2-treated mice exhibited reduced infiltration of tumor-associated M2 macrophages. This was consistent with an increased production of inducible nitric oxide synthase, which is a major enzyme-regulating macrophage inflammatory response to cancer. These data suggest that using an antigalectin 9 mAb with agonistic properties similar to those exerted by galectin-9 may provide a novel multitargeted strategy for the treatment of mesothelioma and possibly other galectin-9 expressing tumors.
RESUMO
Malignant Mesothelioma (MM) is a rare and highly aggressive cancer that develops from mesothelial cells lining the pleura and other internal cavities, and is often associated with asbestos exposure. To date, no effective treatments have been made available for this pathology. Herein, we propose a novel immunotherapeutic approach based on a unique vaccine targeting a series of antigens that we found expressed in different MM tumors, but largely undetectable in normal tissues. This vaccine, that we term p-Tvax, is comprised of a series of immunogenic peptides presented by both MHC-I and -II to generate robust immune responses. The peptides were designed using in silico algorithms that discriminate between highly immunogenic T cell epitopes and other harmful epitopes, such as suppressive regulatory T cell epitopes and autoimmune epitopes. Vaccination of mice with p-Tvax led to antigen-specific immune responses that involved both CD8+ and CD4+ T cells, which exhibited cytolytic activity against MM cells in vitro. In mice carrying MM tumors, p-Tvax increased tumor infiltration of CD4+ T cells. Moreover, combining p-Tvax with an OX40 agonist led to decreased tumor growth and increased survival. Mice treated with this combination immunotherapy displayed higher numbers of tumor-infiltrating CD8+ and CD4+ T cells and reduced T regulatory cells in tumors. Collectively, these data suggest that the combination of p-Tvax with an OX40 agonist could be an effective strategy for MM treatment.
RESUMO
Calpain-2 levels are higher in colorectal tumors resistant to chemotherapy and previous work showed calpain-2 inhibitor therapy reduced inflammation-driven colorectal cancer, but direct effects of the inhibitor on colon cancer cells themselves were not demonstrated. In the present study, five human colon cancer cell lines were directly treated with a calpain-2 inhibitor and results showed increased cell death in 4 of 5 cell lines and decreased anchorage-independent growth for all cell five lines. When tested for levels of calpain-2, three cell lines exhibited increasing levels of this enzyme: HCT15 (low), HCC2998 (medium), and HCT116 (significantly higher). This was consistent with gel shift assays showing that calpain-2 inhibitor reduced of NF-κB nuclear translocation most effectively in HCT116 cells. Ability of calpain-2 inhibitor to impede tumor progression in vivo was evaluated using intrarectal transplant of luciferase-expressing cells for these three cell lines. Results showed that calpain-2 inhibitor therapy reduced tumor growth and increased survival only in mice injected with HCT116 cells. These data suggest calpain-2 inhibitor treatment may be most effective on colorectal tumors expressing highest levels of calpain-2.
Assuntos
Antineoplásicos/farmacologia , Calpaína/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Calpaína/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Progressão da Doença , Regulação para Baixo , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
[This corrects the article DOI: 10.18632/oncotarget.24388.].