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Introduction: Metabolism-associated fatty liver disease (MAFLD) is a global health concern because of its association with obesity, insulin resistance, and other metabolic abnormalities. Methylsulfonylmethane (MSM), an organic sulfur compound found in various plants and animals, exerts antioxidant and anti-inflammatory effects. Here, we aimed to assess the anti-obesity activity and autophagy-related mechanisms of Methylsulfonylmethane. Method: Human hepatoma (HepG2) cells treated with palmitic acid (PA) were used to examine the effects of MSM on autophagic clearance. To evaluate the anti-obesity effect of MSM, male C57/BL6 mice were fed a high-fat diet (HFD; 60% calories) and administered an oral dose of MSM (200 or 400 mg/kg/day). Moreover, we investigated the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin complex 1 (mTORC1)/UNC-51-like autophagy-activating kinase 1 (ULK1) signaling pathway to further determine the underlying action mechanism of MSM. Results: Methylsulfonylmethane treatment significantly mitigated PA-induced protein aggregation in human hepatoma HepG2 cells. Additionally, Methylsulfonylmethane treatment reversed the PA-induced impairment of autophagic flux. Methylsulfonylmethane also enhanced the insulin sensitivity and significantly suppressed the HFD-induced obesity and hepatic steatosis in mice. Western blotting revealed that Methylsulfonylmethane improved ubiquitinated protein clearance in HFD-induced fatty liver. Remarkably, Methylsulfonylmethane promoted the activation of AMPK and ULK1 and inhibited mTOR activity. Conclusion: Our study suggests that MSM ameliorates hepatic steatosis by enhancing the autophagic flux via an AMPK/mTOR/ULK1-dependent signaling pathway. These findings highlight the therapeutic potential of MSM for obesity-related MAFLD treatment.
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BACKGROUND: Obesity, a serious threat to public health, is linked to chronic metabolic complications including insulin resistance, type-2 diabetes, and metabolic dysfunction-associated fatty liver disease (MAFLD). Current obesity medications are challenged by poor effectiveness, poor patient compliance, and potential side effects. Verapamil is an inhibitor of L-type calcium channels, FDA-approved for the treatment of hypertension. We previously investigated the effect of verapamil on modulating autophagy to treat obesity-associated lipotoxicity. This study aims to develop a verapamil transdermal patch and to evaluate its anti-obesity effects. METHODS: Verapamil is loaded in biomimetic vascular bundle-like carboxymethyl pullulan-based supramolecular hydrogel patches cross-linked with citric acid and glycerol linkages (CLCMP). The investigation was then carried out to determine the therapeutic effect of verapamil-loaded CLCMP (Vera@CLCMP) on diet-induced obese mice. RESULTS: Vera@CLCMP hydrogel patches with hierarchically organized and anisotropic pore structures not only improved verapamil bioavailability without modifying its chemical structure but also enhanced verapamil release through the stratum corneum barrier. Vera@CLCMP patches exhibit low toxicity and high effectiveness at delivering verapamil into the systemic circulation through the dermis in a sustained manner. Specifically, transdermal administration of this patch into diet-induced obese mice drastically improved glucose tolerance and insulin sensitivity and alleviated metabolic derangements associated with MAFLD. Furthermore, we uncovered a distinct molecular mechanism underlying the anti-obesity effects associated with the hepatic NLR family pyrin domain-containing 3 (NLRP3) inflammasome and autophagic clearance by the vera@CLCMP hydrogel patches. CONCLUSION: The current study provides promising drug delivery platforms for long-term family treatment of chronic diseases, including obesity and metabolic dysfunctions.
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Chronic exposure to bile acid in the liver due to impaired bile flow induces cholestatic liver disease, resulting in hepatotoxicity and liver fibrosis. Sestrin2, a highly conserved, stress-inducible protein, has been implicated in cellular responses to multiple stress conditions and the maintenance of cellular homeostasis. However, its role in cholestatic liver injury is not fully understood. In this study, we investigated the role of hepatic Sestrin2 in cholestatic liver injury and its underlying mechanisms using in vivo and in vitro approaches. Hepatic Sestrin2 expression was upregulated by activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-ß (C/EBP-ß) after treatment with bile acids and correlated with endoplasmic reticulum (ER) stress responses. Bile-duct ligation (BDL)-induced hepatocellular apoptosis and liver fibrosis were exacerbated in Sestrin2-knockout (Sesn2-/-) mice. Moreover, Sestrin2 deficiency enhanced cholestasis-induced hepatic ER stress, whereas Sestrin2 overexpression ameliorated bile acid-induced ER stress. Notably, the mammalian target of rapamycin (mTOR) inhibitor rapamycin and the AMP-activated protein kinase (AMPK) activator AICAR reversed bile acid-induced ER stress in Sestrin2-deficient cells. Furthermore, Sestrin2 deficiency promoted cholestasis-induced hepatic pyroptosis by activating NLRP3 inflammasomes. Thus, our study provides evidence for the biological significance of Sestrin2 and its relationship with cholestatic liver injury, suggesting the potential role of Sestrin2 in regulating ER stress and inflammasome activation during cholestatic liver injury.
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Colestase , Inflamassomos , Peroxidases , Animais , Colestase/metabolismo , Estresse do Retículo Endoplasmático , Inflamassomos/metabolismo , Fígado/metabolismo , Mamíferos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Peroxidases/genética , Piroptose , Transdução de SinaisRESUMO
Pathological maternal inflammation and abnormal placentation contribute to several pregnancy-related disorders, including preterm birth, intrauterine growth restriction, and preeclampsia. TANK-binding kinase 1 (TBK1), a serine/threonine kinase, has been implicated in the regulation of various physiological processes, including innate immune response, autophagy, and cell growth. However, the relevance of TBK1 in the placental pro-inflammatory environment has not been investigated. In this study, we assessed the effect of TBK1 inhibition on lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation and its underlying mechanisms in human trophoblast cell lines and mouse placenta. TBK1 phosphorylation was upregulated in the trophoblasts and placenta in response to LPS. Pharmacological and genetic inhibition of TBK1 in trophoblasts ameliorated LPS-induced NLRP3 inflammasome activation, placental inflammation, and subsequent interleukin (IL)-1 production. Moreover, maternal administration of amlexanox, a TBK1 inhibitor, reversed LPS-induced adverse pregnancy outcomes. Notably, TBK1 inhibition prevented LPS-induced NLRP3 inflammasome activation by targeting the mammalian target of rapamycin complex 1 (mTORC1). Thus, this study provides evidence for the biological significance of TBK1 in placental inflammation, suggesting that amlexanox may be a potential therapeutic candidate for treating inflammation-associated pregnancy-related complications.
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Inflamassomos/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Complicações na Gravidez/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Trofoblastos/imunologia , Animais , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Placenta/imunologia , Placenta/metabolismo , Gravidez , Complicações na Gravidez/metabolismo , Trofoblastos/metabolismoRESUMO
It has been suggested that oxidative stress involving reactive oxygen species (ROS) induces granulosa cell apoptosis, leading to follicular atresia, and that Tlymphokineactivated killer celloriginated protein kinase (TOPK) suppresses cancer cell apoptosis induced by several stimuli. However, it remains to be determined whether TOPK affects oxidative stressinduced granulosa cell apoptosis. The present study demonstrates that TOPK inhibition increases human granulosa COV434 cell apoptosis induced by hydrogen peroxide (H2O2). Cotreatment with the TOPK inhibitor, OTS514, in combination with H2O2 increased p53 acetylation and its expression, whereas it decreased Sirtuin 1 (SIRT1) expression, contributing to the promotion of apoptosis. In addition, the SIRT1 activator, resveratrol, or the SIRT1 inhibitor, Ex527, reduced or elevated H2O2induced COV434 cell apoptosis, respectively. Furthermore, the p53 inhibitor, Pifithrinµ, diminished the augmentation in poly(ADPribose) polymerase (PARP) cleavage induced by OTS514 plus H2O2, while the Mdm2 antagonist, Nutlin 3, increased PARP cleavage. Moreover, OTS514 further decreased the SIRT1 transcriptional activity decreased by H2O2, but promoted the H2O2induced p53 or p21 transcriptional activity. Notably, the expression of exogenous p53 reduced SIRT1 transcriptional activity. Taken together, the findings of the present study demonstrate that TOPK inhibition promotes p53mediated granulosa cell apoptosis through SIRT1 downregulation in response to H2O2. Therefore, it can be concluded that TOPK suppresses H2O2induced apoptosis through the modulation of the p53/SIRT1 axis, suggesting a potential role of TOPK in the regulation of human granulosa cell apoptosis, leading to the promotion of abnormal follicular development.
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Apoptose/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Atresia Folicular/efeitos dos fármacos , Atresia Folicular/metabolismo , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Oncogenic activation of the mammalian target of rapamycin complex 1 (mTORC1) leads to endometrial cancer cell growth and proliferation. Sestrin2 (SESN2), a highly conserved stress-inducible protein, is involved in homeostatic regulation via inhibition of reactive oxygen species (ROS) and mTORC1. However, the role of SESN2 in human endometrial cancer remains to be investigated. Here, we investigated expression, clinical significance, and underlying mechanisms of SESN2 in endometrial cancer. SESN2 was upregulated more in endometrial cancer tissues than in normal endometrial tissues. Furthermore, upregulation of SESN2 statistically correlated with shorter overall survival and disease-free survival in patients with endometrial cancer. SESN2 expression strongly correlated with mTORC1 activity, suggesting its impact on prognosis in endometrial cancer. Additionally, knockdown of SESN2 promoted cell proliferation, migration, and ROS production in endometrial cancer cell lines HEC-1A and Ishikawa. Treatment of these cells with mTOR inhibitors reversed endometrial cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) marker expression. Moreover, in a xenograft nude mice model, endometrial cancer growth increased by SESN2 knockdown. Thus, our study provides evidence for the prognostic significance of SESN2, and a relationship between SESN2, the mTORC1 pathway, and endometrial cancer growth, suggesting SESN2 as a potential therapeutic target in endometrial cancer.
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Emerging evidence indicates that aberrant maternal inflammation is associated with several pregnancy-related disorders such as preeclampsia, preterm birth, and intrauterine growth restriction. Sirtuin1 (SIRT1), a class III histone deacetylase, is involved in the regulation of various physiopathological processes including cellular inflammation and metabolism. However, the effect of SIRT1 on the placental proinflammatory environment remains to be elucidated. In this study, we investigated the effect of SIRT1 on lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation and its underlying mechanisms in human first-trimester trophoblasts (Sw.71 and HTR-8/SVneo cells). Treatment with LPS elevated SIRT1 expression and induced NLRP3 inflammasome activation in mouse placental tissues and human trophoblasts. Knockdown of SIRT1 enhanced LPS-induced NLRP3 inflammasome activation, inflammatory signaling, and subsequent interleukin (IL)-1ß secretion. Furthermore, knockdown of NLRP3 considerably attenuated the increase of IL-1ß secretion in SIRT1-knockdown cells treated with LPS. Moreover, SIRT1 inhibited LPS-induced NLRP3 inflammasome activation by reducing oxidative stress. This study revealed a novel mechanism via which SIRT1 exerts anti-inflammatory effects, suggesting that SIRT1 is a potential therapeutic target for the prevention of inflammation-associated pregnancy-related complications.
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Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sirtuína 1/genética , Trofoblastos/metabolismo , Animais , Feminino , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nascimento Prematuro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismoRESUMO
PROBLEM: Maternal obesity induces elevated saturated fatty acid palmitate levels in the blood and causes pregnancy complications such as gestational diabetes, preeclampsia, fetal growth abnormalities, and stillbirth. Sestrin2, a highly conserved stress-inducible protein, is involved in the cellular responses of various stress conditions and homeostatic regulation. However, the effects of Sestrin2 on trophoblast cells have not yet been investigated. Here, we investigated the role of Sestrin2 in palmitate-induced lipotoxicity and its underlying mechanisms in human first-trimester trophoblast cells (Sw.71). METHOD OF STUDY: Mouse placental tissues were obtained from low-fat diet-fed mice (n = 14) and high-fat diet-fed mice (n = 14) at gestation day 17.5. Sw.71 cells were treated with palmitate or bovine serum albumin as vehicle controls. The role of Sestrin2 in palmitate-induced lipotoxicity was examined by immunocytochemistry, immunoblot analysis, quantitative real-time PCR, and invasion assay. RESULTS: Expression of placental Sestrin2 was elevated in high-fat diet-fed dams compared to that of low-fat diet-fed dams. Prolonged treatment of Sw.71 cells with palmitate-induced endoplasmic reticulum (ER) stress-dependent expressions of Sestrin2 protein and mRNA, and the treatment also triggered apoptosis. Knockdown of Sestrin2 increased palmitate-mediated ER stress, inflammatory signaling, and apoptosis. Furthermore, Sestrin2 suppressed impaired trophoblast invasion caused by palmitate and attenuated palmitate-induced ER stress and inflammation via AMPK/mTORC1 pathways. CONCLUSION: Our study provides the relationship between Sestrin2, AMPK/mTORC1 pathway, and trophoblast function, suggesting that Sestrin2 may be a novel potential therapeutic target for the prevention of pregnancy complications.
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Obesidade/metabolismo , Peroxidases/metabolismo , Complicações na Gravidez/metabolismo , Trofoblastos/metabolismo , Adenilato Quinase/metabolismo , Animais , Apoptose , Movimento Celular , Células Cultivadas , Estresse do Retículo Endoplasmático , Feminino , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Palmitatos/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Transdução de Sinais , Trofoblastos/patologiaRESUMO
Sirt1, also known as the longevity gene, is an NAD+-dependent class III histone deacetylase that has been extensively studied in multiple areas of research including cellular metabolism, longevity, cancer, autoimmunity, and immunity. However, little is known about the function of Sirt1 in B cells. This study aimed to investigate the role of Sirt1 in the expression pattern of mRNAs in the resting B cells of mice. CD19+ B cell-specific inducible Sirt1 knockout (KO) mice were divided into tamoxifen-treated Sirt1 KO group (S19T) or control group (S19). mRNAs extracted from resting B cells of both groups were analyzed for differentially expressed genes (DEG) using microarray. DEG analysis showed significant differential expression of 20 genes, of which Hspa1a and Hspa1b showed the highest fold change (FC) in S19T compared with S19 (p value < 0.01 and FC > 3). Further, Kyoto Encyclopedia of Genes and Genomes analysis identified pathways associated with diseases, organismal systems, and antigen processing and presentation. Additionally, the pathways known to involve Hspa1a and Hspa1b were also activated in the S19T group. On the other hand, after in vitro stimulation with lipopolysaccharide, cell viability and IgM production were significantly decreased in Sirt1 KO B cells, while expressions of TNF-α, IL-6, and IL-10 were increased. In summary, our study reveals that Sirt1 may maintain the quiescent state in resting B cells by suppressing the increase of Hspa1a and Hspa1b. This work provides a foundation for further studies on the functional roles of Sirt1 in B cells.
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Linfócitos B/metabolismo , Proteínas de Choque Térmico HSP70/genética , Sirtuína 1/deficiência , Animais , Linfócitos B/fisiologia , Sobrevivência Celular , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
Obesity and overweight, the most serious health problems, are associated with chronic metabolic complications such as type 2 diabetes, insulin resistance, and nonalcoholic fatty liver disease (NAFLD). However, current pharmacological therapies for obesity are challenged by potential side effects, low effectiveness, and low aqueous solubility, which limit their clinical application. Here, we develop nifedipine-loaded nanoparticles (NFD-NPs) that alleviate obesity-related metabolic dysfunction to be used as instruments for translational medicine. Nanoparticles (NPs) composed of poly (lactic-co-glycolic acid) (PLGA) not only enhance water solubility of hydrophobic nifedipine (NFD), a calcium channel blocker, without modifying the chemical structure of NFD for intravenous administration, but also allow prolonged release of NFD in vivo. NFD-NPs do not show cytotoxicity and reduce palmitate-induced protein inclusions and endoplasmic reticulum stress in human hepatoma HepG2 cells. Importantly, tail-vein injection of NFD-NPs into diet-induced obese mice results in sustained retention of NFD-NPs in the liver and suppression of metabolic derangements associated with NAFLD by enhancing autophagic clearance through Ca2+/calmodulin-dependent kinase II (CaMKII) phosphorylation, consequently decreasing diet-induced insulin resistance and improving glucose tolerance. Our findings offer new clinical tools for NP-mediated pharmaceutical strategies to treat NAFLD and its related metabolic dysfunction.
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Autofagia/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/uso terapêutico , Nifedipino/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Administração Intravenosa , Animais , Bloqueadores dos Canais de Cálcio/administração & dosagem , Preparações de Ação Retardada/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Hep G2 , Humanos , Resistência à Insulina , Masculino , Camundongos Endogâmicos C57BL , Nifedipino/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/químicaRESUMO
Red emitting europium (III) complexes Eu(TFAAN)3(P(Oct)3)3 (TFAAN = 2-(4,4,4-Trifluoroacetoacetyl)naphthalene, P(Oct)3 = trioctylphosphine) chelated on carboxymethyl dextran coated superparamagnetic iron oxide nanoparticles (CMD-SPIONs) was synthesized and the step wise synthetic process was reported. All the excitation spectra of distinctive photoluminesces were originated from f-f transition of EuIII with a strong red emission. The emission peaks are due to the hypersensitive transition 5D0â7F2 at 621 nm and 5D0â7F1 at 597 nm, 5D0â7F0 at 584 nm. No significant change in PL properties due to addition of CMD-SPIONs was observed. The cytotoxic effects of different concentrations and incubation times of Eu(TFAAN)3(P(Oct)3)3 chelated CMD-SPIONs were evaluated in HEK293T and HepG2 cells using the WST assay. The results imply that Eu(TFAAN)3(P(Oct)3)3 chelated CMD-SPIONs are not affecting the cell viability without altering the apoptosis and necrosis in the range of 10 to 240 µg/mL concentrations.
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Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella), a virulent pathogen, is known to induce hostcell death. Using reverse transcriptionquantitative polymerase chain reaction, a 28fold increase of microRNA (miR)155 expression in RAW 264.7 macrophages was observed following infection with Salmonella for 24 h. This miR155 upregulation increased macrophage cell death by up to 40% in 48 h following infection. Western blot analysis revealed that receptor interacting protein 1 (RIP1) and 3 (RIP3) were increased at 18 h following miR155 transfection to macrophages, similar to Salmonella infection. In addition, inhibition of RIP1 by preincubating macrophages with necrostatin1, a RIP1 specific inhibitor, increased the viability of Salmonellainfected cells and miR155transfected cells by up to 20%. The cleavage of poly (adenosine diphosphateribose) polymerase1 (PARP1) was also enhanced by miR155 induction upon Salmonella infection. Therefore, it was suggested that RIP1/3induced necroptosis and PARP1mediated necrosis caused by miR155 induction may represent distinct routes of programmed necrotic cell death of Salmonellainfected macrophages.
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Proteínas Ativadoras de GTPase/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , MicroRNAs/genética , Interferência de RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Salmonella typhimurium/fisiologia , Animais , Morte Celular/genética , Regulação da Expressão Gênica , Camundongos , Necrose/genética , Células RAW 264.7 , Infecções por Salmonella/genética , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologiaRESUMO
Chronic exposure to hydrophobic bile acids such as chenodeoxycholic acid (CDCA) and cholic acid (CA) in the liver during cholestasis causes hepatotoxicity and inflammatory response. However, the detailed mechanisms regarding the role of autophagy in cholestatic hepatotoxicity remain largely unknown. Here we determined autophagic clearance in livers of bile duct-ligated mice, in which bile acids accumulate, and in human hepatoma HepG2 cells treated with CDCA and CA. The accumulation of bile acids caused defective autophagic clearance, shown by the accumulation of insoluble p62 and ubiquitinated proteins and cell death accompanied by caspase-3 processing. Hepatocytes exposed to bile acids also showed the accumulation of autophagosomes via suppressed autophagy flux. Treatment of CDCA markedly suppressed Beclin-1 expression, which exhibits a higher cytotoxicity than CA. Moreover, pharmacological or genetic inhibition of autophagy enhanced bile acid-induced cell death. Finally, in vivo, bile duct ligation led to aberrant accumulation of p62 and ubiquitinated proteins in the liver. Our data demonstrate that inhibited autophagy is an essential component of liver injury during cholestasis.
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Autofagia , Ácidos e Sais Biliares/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Fígado/metabolismo , Proteínas Ubiquitinadas/biossíntese , Animais , Células Hep G2 , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ubiquitinação , Regulação para CimaRESUMO
Dysregulated serum fatty acids are associated with a lipotoxic placental environment, which contributes to increased pregnancy complications via altered trophoblast invasion. However, the role of saturated and unsaturated fatty acids in trophoblastic autophagy has yet to be explored. Here, we demonstrated that prolonged exposure of saturated fatty acids interferes with the invasiveness of human extravillous trophoblasts. Saturated fatty acids (but not unsaturated fatty acids) inhibited the fusion of autophagosomes and lysosomes, resulting in the formation of intracellular protein aggregates. Furthermore, when the trophoblast cells were exposed to saturated fatty acids, unsaturated fatty acids counteracted the effects of saturated fatty acids by increasing degradation of autophagic vacuoles. Saturated fatty acids reduced the levels of the matrix metalloproteinases (MMP)-2 and MMP-9, while unsaturated fatty acids maintained their levels. In conclusion, saturated fatty acids induced decreased trophoblast invasion, of which autophagy dysfunction plays a major role.
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Autofagia/imunologia , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Trofoblastos/imunologia , Autofagossomos/imunologia , Autofagossomos/metabolismo , Linhagem Celular , Movimento Celular/imunologia , Ácidos Graxos/imunologia , Ácidos Graxos Insaturados/imunologia , Feminino , Humanos , Lisossomos/imunologia , Lisossomos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Obesidade/imunologia , Obesidade/metabolismo , Gravidez , Complicações na Gravidez/imunologia , Complicações na Gravidez/metabolismo , Agregação Patológica de Proteínas/imunologia , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.
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Células-Tronco Embrionárias Murinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Inativação do Cromossomo X/genética , Animais , Técnicas de Cultura de Células , Feminino , Camundongos , Proteínas Mutantes/metabolismoRESUMO
Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.
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Regulação da Expressão Gênica no Desenvolvimento , Genes Ligados ao Cromossomo X , Ubiquitina-Proteína Ligases/metabolismo , Animais , Camundongos , RNA Longo não Codificante/metabolismo , Análise de Sequência de RNA , Inativação do Cromossomo XRESUMO
In female mice, two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X chromosome. Later, around implantation, epiblast cells of the inner cell mass that give rise to the embryo reactivate the paternal X chromosome and undergo a random form of XCI (rXCI). Xist, a long non-coding RNA crucial for both forms of XCI, is activated by the ubiquitin ligase RLIM (also known as Rnf12). Although RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here we show that RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking RLIM from pre-implantation stages onwards show hallmarks of XCI, including Xist clouds and H3K27me3 foci, and have full embryogenic potential. These results provide evidence that RLIM is dispensable for rXCI, indicating that in mice an RLIM-independent mechanism activates Xist in the embryo proper.
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Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Inativação do Cromossomo X/genética , Animais , Regulação para Baixo , Implantação do Embrião , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Histonas/química , Histonas/metabolismo , Hibridização in Situ Fluorescente , Lisina/metabolismo , Metilação , Camundongos , Camundongos Knockout , RNA Longo não Codificante/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The X-linked gene Rnf12 encodes the ubiquitin ligase really interesting new gene (RING) finger LIM domain-interacting protein (RLIM)/RING finger protein 12 (Rnf12), which serves as a major sex-specific epigenetic regulator of female mouse nurturing tissues. Early during embryogenesis, RLIM/Rnf12 expressed from the maternal allele is crucial for the development of extraembryonic trophoblast cells. In contrast, in mammary glands of pregnant and lactating adult females RLIM/Rnf12 expressed from the paternal allele functions as a critical survival factor for milk-producing alveolar cells. Although RLIM/Rnf12 is detected mostly in the nucleus, little is known about how and in which cellular compartment(s) RLIM/Rnf12 mediates its biological functions. Here we demonstrate that RLIM/Rnf12 protein shuttles between nucleus and cytoplasm and this is regulated by phosphorylation of serine S214 located within its nuclear localization sequence. We show that shuttling is important for RLIM to exert its biological functions, as alveolar cell survival activity is inhibited in cells expressing shuttling-deficient nuclear or cytoplasmic RLIM/Rnf12. Thus regulated nucleocytoplasmic shuttling of RLIM/Rnf12 coordinates cellular compartments during mammary alveolar cell survival.
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Transporte Ativo do Núcleo Celular/genética , Sobrevivência Celular/genética , Glândulas Mamárias Animais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Núcleo Celular/genética , Desenvolvimento Embrionário , Epigênese Genética/genética , Feminino , Células HeLa , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Fosforilação , Gravidez , Processos de Determinação Sexual , Ubiquitina-Proteína Ligases/genéticaRESUMO
In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), whereas extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12, which encodes the ubiquitin ligase Rnf12/RLIM. We find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy, with alveolar cells that lack RLIM undergoing apoptosis as they begin to differentiate. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom maternal XCI in mammary epithelial cells. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and, together with population models, reveal implications of transgenerational epigenetic inheritance.
Assuntos
Sobrevivência Celular , Glândulas Mamárias Animais/citologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Epigênese Genética , Feminino , Impressão Genômica , Masculino , Glândulas Mamárias Animais/fisiologia , Camundongos , Gravidez , Ubiquitina-Proteína Ligases/genética , Inativação do Cromossomo XRESUMO
Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.