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BACKGROUND/AIM: The small intestine is one of the organs most vulnerable to ionizing radiation (IR) damage. However, methods to protect against IR-induced intestinal injury are limited. CBLB502, a Toll-like receptor 5 (TLR5) agonist from Salmonella flagellin, exerts radioprotective effects on various tissues and organs. However, the molecular mechanisms by which CBLB502 protects against IR-induced intestinal injury remain unclear. Thus, this study aimed to elucidate the mechanisms underlying IR-induced intestinal injury and the protective effects of CBLB502 against this condition in mice. MATERIALS AND METHODS: Mice were administered 0.2 mg/kg CBLB502 before IR at different doses for different time points, and then the survival rate, body weight, hemogram, and histopathology of the mice were analyzed. RESULTS: CBLB502 reduced IR-induced intestinal injury. RNA-seq analysis revealed that different doses and durations of IR induced different regulatory patterns. CBLB502 protected against intestinal injury mainly after IR by reversing the expression of IR-induced genes and regulating immune processes and metabolic pathways. CONCLUSION: This study preliminarily describes the regulatory mechanism of IR-induced intestinal injury and the potential molecular protective mechanism of CBLB502, providing a basis for identifying the functional genes and molecular mechanisms that mediate protection against IR-induced injury.
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Protectores contra Radiación , Animales , Ratones , Protectores contra Radiación/farmacología , Receptor Toll-Like 5/agonistas , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismo , Masculino , Radiación Ionizante , Receptores Toll-Like/metabolismo , Receptores Toll-Like/agonistas , Traumatismos por Radiación/tratamiento farmacológico , Traumatismos por Radiación/patología , Intestinos/efectos de los fármacos , Intestinos/patología , Intestinos/efectos de la radiación , Modelos Animales de Enfermedad , Agonistas de los Receptores Toll-Like , PéptidosRESUMEN
Ferroptosis, a recently discovered type of programmed cell death triggered by excessive accumulation of irondependent lipid peroxidation, is linked to several malignancies, including nonsmall cell lung cancer. Long noncoding RNAs (lncRNAs) are involved in ferroptosis; however, data on their role and mechanism in cancer therapy remains limited. Therefore, the aim of the present study was to identify ferroptosisassociated mRNAs and lncRNAs in A549 lung cancer cells treated with RASselective lethal 3 (RSL3) and ferrostatin1 (Fer1) using RNA sequencing. The results demonstrated that lncRNA lung cancerassociated transcript 1 (LUCAT1) was significantly upregulated in lung adenocarcinoma and lung squamous cell carcinoma tissues. Coexpression analysis of differentially expressed mRNAs and lncRNAs suggested that LUCAT1 has a crucial role in ferroptosis. LUCAT1 expression was markedly elevated in A549 cells treated with RSL3, which was prevented by coincubation with Fer1. Functionally, overexpression of LUCAT1 facilitated cell proliferation and reduced the occurrence of ferroptosis induced by RSL3 and Erastin, while inhibition of LUCAT1 expression reduced cell proliferation and increased ferroptosis. Mechanistically, downregulation of LUCAT1 resulted in the downregulation of both GTP cyclohydrolase 1 (GCH1) and ferroptosis suppressor protein 1 (FSP1). Furthermore, inhibition of LUCAT1 expression upregulated microRNA (miR)34a5p and then downregulated GCH1. These results indicated that inhibition of LUCAT1 expression promoted ferroptosis by modulating the downregulation of GCH1, mediated by miR34a5p. Therefore, the combination of knocking down LUCAT1 expression with ferroptosis inducers may be a promising strategy for lung cancer treatment.
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Regulación hacia Abajo , Ferroptosis , GTP Ciclohidrolasa , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , MicroARNs , ARN Largo no Codificante , Humanos , Ferroptosis/genética , MicroARNs/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células A549 , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Proliferación Celular , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Masculino , Línea Celular Tumoral , Femenino , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismoRESUMEN
Ionizing radiation (IR) induces severe hematopoietic injury by causing DNA and RNA damage as well as activating the immune responses, necessitating the development of effective therapeutic strategies. Ribonuclease L (RNase L) as an innate immune response pathway is triggered by exogenous and endogenous abnormal dsRNA under viral infection and dyshomeostasis, thereby activating the immune responses. Thus, we investigated the effect of RNase L on irradiation-induced bone marrow damage using RNase L knockout (RNase L-/-) mice. Phenotypic analysis revealed that RNase L knockout mitigates irradiation-induced injury in the bone marrow. Further investigation into the mechanism of RNase L by RNA-seq, qRT-PCR, and CBA analysis demonstrated that RNase L deficiency counteracts the upregulation of genes related to immune responses induced by irradiation, including cytokines and interferon-stimulated genes. Moreover, RNase L deficiency inhibits the increased levels of immunoglobulins in serum induced by irradiation. These findings indicate that RNase L plays a role in the immune response induced by irradiation in the bone marrow. This study further enhances our understanding of the biological functions of RNase L in the immune response induced by irradiation and offers a novel approach for managing irradiation-induced bone marrow injury through the regulation of RNase L activation.
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Médula Ósea , Inmunidad Innata , Ratones , Animales , Médula Ósea/metabolismo , Ratones Noqueados , Ratones Endogámicos CBA , ARN Bicatenario , Endorribonucleasas/metabolismoRESUMEN
BRISC (BRCC3 isopeptidase complex) is a deubiquitinating enzyme that has been linked with inflammatory processes, but its role in liver diseases and the underlying mechanism are unknown. Here, we investigated the pathophysiological role of BRISC in acute liver failure using a mice model induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS). We found that the expression of BRISC components was dramatically increased in kupffer cells (KCs) upon LPS treatment in vitro or by the injection of LPS in D-GalN-sensitized mice. D-GalN plus LPS-induced liver damage and mortality in global BRISC-null mice were markedly attenuated, which was accompanied by impaired hepatocyte death and hepatic inflammation response. Constantly, treatment with thiolutin, a potent BRISC inhibitor, remarkably alleviated D-GalN/LPS-induced liver injury in mice. By using bone marrow-reconstituted chimeric mice and cell-specific BRISC-deficient mice, we demonstrated that KCs are the key effector cells responsible for protection against D-GalN/LPS-induced liver injury in BRISC-deficient mice. Mechanistically, we found that hepatic and circulating levels of TNF-α, IL-6, MCP-1, and IL-1ß, as well as TNF-α- and MCP-1-producing KCs, in BRISC-deleted mice were dramatically decreased as early as 1 h after D-GalN/LPS challenge, which occurred prior to the elevation of the liver injury markers. Moreover, LPS-induced proinflammatory cytokines production in KCs was significantly diminished by BRISC deficiency in vitro, which was accompanied by potently attenuated NF-κB activation. Restoration of NF-κB activation by two small molecular activators of NF-κB p65 effectively reversed the suppression of cytokines production in ABRO1-deficient KCs by LPS. In conclusion, BRISC is required for optimal activation of NF-κB-mediated proinflammatory cytokines production in LPS-treated KCs and contributes to acute liver injury. This study opens the possibility to develop new strategies for the inhibition of KCs-driven inflammation in liver diseases.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Animales , Ratones , FN-kappa B/metabolismo , Lipopolisacáridos/farmacología , Macrófagos del Hígado/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Hígado/metabolismo , Inflamación/metabolismo , Galactosamina , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismoRESUMEN
BACKGROUND/AIM: CBLB502, a Toll-like receptor-5 agonist derived from Salmonella flagellin, exerts protective roles against irradiation and chemical drugs in mammalian tissues and stimulates tissue regeneration. This study aimed to investigate whether CBLB502 can protect against liver and kidney damage induced by the chemotherapeutic drug cisplatin (CDDP) and the underlying mechanism of the protective effect. MATERIALS AND METHODS: Mice were pretreated with CBLB502 [0.2 mg/kg, intraperitoneal (i.p.) injection] 0.5 h prior to administration of CDDP (20 mg/kg, i.p. injection), and analyses of the liver and kidney indices, blood biochemistry, and histopathology were performed. RESULTS: Pretreatment with CBLB502 alleviated CDDP-induced liver and kidney damage. RNA sequencing and bioinformatic analysis indicated that CDDP induced a similar damage-promoting gene regulation pattern in the liver and kidney. CBLB502 protected against liver and kidney damage only after CDDP treatment primarily via different pathways. However, some CBLB502-regulated genes were common between the liver and kidney, including those involved in blood coagulation, fibrinolysis, hemostasis, apoptotic regulation, NF-kappaB signaling, and response to lipopolysaccharide, suggesting a general protective effect by CBLB502. CONCLUSION: Our data provide insights into the protective mechanism of CBLB502 against CDDP-induced tissue damage in the liver and kidney and might provide a basis for future studies on functional genes and regulatory mechanisms that mediate protection against chemoradiotherapy-induced damage.
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Cisplatino , Hígado , Animales , Ratones , Cisplatino/efectos adversos , Adyuvantes Inmunológicos , Riñón , Receptores Toll-Like , MamíferosRESUMEN
Hydroxyl radical (·OH) generated by the Fenton reaction between transition metal ions and hydrogen peroxide (H2O2) can induce significant cellular damage. However, the specific mechanism of ·OH-induced cell death has not been systematically studied. In this study, we reacted FeSO4 and Fe3O4 magnetic nanoparticles with H2O2 and found that ·OH generated from the intracellular Fenton reaction can lead to significant cell death. The Fenton reaction between Fe2+ with H2O2 resulted in a shift in lipid peroxidation and cell cycle arrest. It is noteworthy that the ·OH generated from the Fenton reaction triggered severe apoptosis but did not lead to DNA double-strand breakage. Our results suggest that the Fenton reaction had acute cytotoxicity, which was primarily due to ·OH produced from the Fenton reaction inducing lipid peroxidation and apoptosis and modulating the cell cycle, but not by inducing DNA damage.
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Peróxido de Hidrógeno , Hierro , Peroxidación de Lípido , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Radical Hidroxilo , ADN/metabolismo , Ciclo Celular , ApoptosisRESUMEN
Patients with gastric cancer (GC) are usually first diagnosed at an advanced stage due to the absence of obvious symptoms at an early GC (EGC) stage. Therefore, it is necessary to identify an effective screening method to detect precursor lesions of GC (PLGC) and EGC to increase the 5-year survival rate of patients. Cell-free RNA, as a biomarker, has shown potential in early diagnosis, personalised treatment, and prognosis of cancer. In this study, six RNAs (CEBPA-AS1, INHBA-AS1, AK001058, UCA1, PPBP, and RGS18) were analysed via real-time quantitative polymerase chain reaction (RT-qPCR) using the plasma of patients with EGC and PLGC to identify diagnostic biomarkers. The receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic accuracy. Among the six RNAs, four lncRNAs (CEBPA-AS1, INHBA-AS1, AK001058, and UCA1) were upregulated and two mRNAs (PPBP and RGS18) were downregulated in the plasma of patients with PLGC and EGC. According to the findings of the ROC analysis, the four-RNA combination of INHBA-AS1, AK001058, UCA1, and RGS18 had the highest area under the curve (AUC) value for determining risk of GC in patients with PLGC and the six-RNA combination including CEBPA-AS1, INHBA-AS1, AK001058, UCA1, PPBP, and RGS18 had the highest AUC value for determining the risk of GC in patients with EGC. The results suggest the potential usefulness of noninvasive biomarkers for the molecular diagnosis of GC at earlier stages.
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Pyroptosis is mediated by gasdermins and serves a critical role in ionizing radiation (IR)induced damage in normal tissues, but its role in cancer radiotherapy and underlying mechanisms remains unclear. Long noncoding (lnc) RNAs serve important roles in regulating the radiosensitivity of cancer cells. The present study aimed to investigate the mechanistic involvement of lncRNAs in IRinduced pyroptosis in human colorectal cancer HCT116 cells. LncRNA, microRNA (miR)448 and gasdermin E (GSDME) levels were evaluated using reverse transcriptionquantitative polymerase chain reaction. Protein expression and activation of gasdermins were measured using western blotting. The binding association between miR448 and GSDME was assessed using the dualluciferase reporter assay. Pyroptosis was examined using phasecontrast microscopy, flow cytometry, Cell Counting Kit8 assay and lactate dehydrogenase release assay. IR dosedependently induced GSDMEmediated pyroptosis in HCT116 cells. GSDME was identified as a downstream target of miR448. LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) was upregulated in response to IR and enhanced GSDME expression by negatively regulating miR448 expression. Notably, NEAT1 knockdown suppressed IRinduced pyroptosis, fulllength GSDME expression and GSDME cleavage compared with that in irradiated cells. In addition, NEAT1 knockdown rescued the IRinduced decrease in cell viability in HCT116 cells. The findings of the present study indicated that lncRNA NEAT1 modulates IRinduced pyroptosis and viability in HCT116 cells via miR448 by regulating the expression, but not activation of GSDME. The present study provides crucial mechanistic insight into the potential role of lncRNA NEAT1 in IRinduced pyroptosis.
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Neoplasias Colorrectales/patología , MicroARNs/fisiología , Piroptosis/efectos de la radiación , ARN Largo no Codificante/fisiología , Receptores de Estrógenos/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/radioterapia , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Tolerancia a Radiación , Receptores de Estrógenos/fisiologíaRESUMEN
Long-term exposures to low-dose radiation (LDR) may trigger several specific biological responses, including dysregulation of the immune and inflammatory systems. Here, we examined whether biodosimetry of LDR can be used to protect tissues from radiation or assess cancer risk. Mice were subjected to gamma-irradiation with repeated or single-dose LDR, and then the organ indices, peripheral hemogram, and blood biochemistry were analyzed. An antibody array was applied followed by enzyme-linked immunosorbent assay to evaluate the utility of multiple plasma proteins as biomarkers of repeated LDR in a murine model. LDR induced inapparent symptoms but slight variations in peripheral blood cell counts and alterations in blood biochemical indicator levels. Specific plasma proteins in the LDR groups were altered in response to a higher dose of irradiation at the same time points or a single-dose equivalent to the same total dose. Plasma levels of interleukin (IL)-5, IL-12p40, P-selectin, and serum amyloid A1 were associated with the LDR dose and thus may be useful as dosimetric predictors of LDR in mice. Estimating the levels of certain plasma proteins may yield promising biodosimetry parameters to accurately identify individuals exposed to LDR, facilitating risk assessment of long-term LDR exposure in individuals.
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Pharmacologically inhibiting nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome activation results in potent therapeutic effects in a wide variety of preclinical inflammatory disease models. NLRP3 deubiquitination is essential for efficient NLRP3 inflammasome activity, but it remains unclear whether this process can be harnessed for therapeutic benefit. Here, we show that thiolutin (THL), an inhibitor of the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease, blocks NLRP3 inflammasome activation by canonical, noncanonical, alternative, and transcription-independent pathways at nanomolar concentrations. In addition, THL potently inhibited the activation of multiple NLRP3 mutants linked with cryopyrin-associated periodic syndromes (CAPS). Treatment with THL alleviated NLRP3-related diseases in mouse models of lipopolysaccharide-induced sepsis, monosodium urate-induced peritonitis, experimental autoimmune encephalomyelitis, CAPS, and methionine-choline-deficient diet-induced nonalcoholic fatty liver disease. Mechanistic studies revealed that THL inhibits the BRCC3-containing isopeptidase complex (BRISC)-mediated NLRP3 deubiquitination and activation. In addition, we show that holomycin, a natural methyl derivative of THL, displays an even higher inhibitory activity against NLRP3 inflammasome than THL. Our study validates that posttranslational modification of NLRP3 can be pharmacologically targeted to prevent or treat NLRP3-associated inflammatory diseases. Future clinical development of derivatives of THL may provide new therapies for NLRP3-related diseases.
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Enzimas Desubicuitinizantes/antagonistas & inhibidores , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Animales , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Sangre Fetal , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lactamas/farmacología , Lactamas/uso terapéutico , Lipopolisacáridos , Masculino , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/inmunología , Embarazo , Cultivo Primario de Células , Pirrolidinonas/farmacología , Pirrolidinonas/uso terapéutico , Células THP-1 , Ubiquitinación/efectos de los fármacosRESUMEN
BACKGROUND: Toll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating the hepatic immune response and show hepatoprotective effects in mouse models of hepatic diseases. However, the role of TLR5 in experimental models of liver regeneration has not been reported. This study aimed to investigate the role of TLR5 in partial hepatectomy (PHx)-induced liver regeneration. METHODS: We performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice, as a model of liver regeneration. Bacterial flagellin content was measured with ELISA, and hepatic TLR5 expression was determined with quantitative PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, we analyzed bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression with immunohistochemistry (IHC) staining. The effects of TLR5 during the priming phase of liver regeneration were examined with quantitative PCR analyses of immediate early gene mRNA levels, and with Western blotting analysis of hepatic NF-κB and STAT3 activation. Cytokine and growth factor production after PHx were detected with real-time PCR and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx. RESULTS: The bacterial flagellin content in the serum and liver increased, and the hepatic TLR5 expression was significantly up-regulated in WT mice after PHx. TLR5-deficient mice exhibited diminished numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokine and growth factor production. Moreover, PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5-/- mice, as compared with WT mice. Consistently, the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation, which was correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver. Furthermore, Tlr5-/- mice displayed significantly lower hepatic lipid concentrations and smaller Oil Red O positive areas than those in control mice after PHx. CONCLUSION: We reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx. Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest the potential of TLR5 agonist to promote liver regeneration.
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Regeneración Hepática/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 5/uso terapéutico , Animales , Modelos Animales de Enfermedad , Regeneración Hepática/fisiología , Ratones , Ratones Endogámicos C57BL , Estadísticas no Paramétricas , Receptor Toll-Like 5/metabolismoRESUMEN
Abraxas brother protein 1 (ABRO1) is a subunit of the deubiquitinating enzyme BRCC36-containing isopeptidase complex and plays important roles in cellular responses to stress by interacting with its binding partners, such as ubiquitin-specific peptidase 7, p53, activating transcription factor 4, THAP-domain containing 5, and serine hydroxymethyltransferase. However, the transcriptional regulation of ABRO1 remains unexplored. In this study, we identified and characterized the core regulatory elements of the human ABRO1 gene and mapped them to the ABRO1 promoter region. Additionally, 5' rapid amplification of cDNA ends revealed that the transcriptional start site (TSS) was located -13 bp upstream from the start codon. Reporter gene, chromatin immunoprecipitation, and electrophoretic mobility shift assays demonstrated that ABRO1 transcription was regulated through cis-acting elements located in the region -89 to -59 bp upstream of the ABRO1 TSS and that these elements were targeted by yin yang 1 transcription factor (YY1). Moreover, YY1 overexpression increased human ABRO1 mRNA and protein expression, and small-interfering RNA-mediated downregulation of YY1 attenuated ABRO1 expression. These results suggested that YY1 positively regulated human ABRO1 expression by binding to cis-acting elements located in the ABRO1 TSS.
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Proteínas Asociadas a Matriz Nuclear/genética , Proteasas Ubiquitina-Específicas/genética , Factor de Transcripción YY1/metabolismo , Animales , Línea Celular , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Matriz Nuclear/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
BRCA1/BRCA2-containing complex subunit 3 (BRCC3) is a lysine 63-specific deubiquitinase involved in multiple biological processes, such as DNA repair and immune responses. However, the regulation mechanism for BRCC3 protein stability is still unknown. Here, we demonstrate that BRCC3 is mainly degraded through the ubiquitin-proteasome pathway. The HECT-type E3 ubiquitin ligase WWP2 modulates BRCC3 ubiquitination and degradation. ABRO1, a subunit of the BRCC36 isopeptidase complex (BRISC), competes with WWP2 to bind to BRCC3, thereby preventing WWP2-mediated BRCC3 ubiquitination and enhancing BRCC3 stability. Functionally, we show that lentivirus-mediated overexpression of WWP2 in murine macrophages inhibits NLRP3 inflammasome activation by decreasing BRCC3 protein level. This study provides the first insights into the regulation of BRCC3 stability and expands our knowledge about the physiological function of WWP2.
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Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Línea Celular , Células Cultivadas , Enzimas Desubicuitinizantes/genética , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Proteínas Asociadas a Matriz Nuclear/genética , Estabilidad Proteica , Proteolisis , Proteasas Ubiquitina-Específicas/genética , UbiquitinaciónRESUMEN
High-dose ionizing radiation (IR) alters the expression levels of non-coding RNAs (ncRNAs). However, the roles of ncRNAs and mRNAs in mediating radiation protection by radioprotectants remain unknown. Microarrays were used to determine microRNA (miRNA), long ncRNA (lncRNA), and mRNA expression profiles in the bone marrow of irradiated mice pretreated with amifostine, CBLB502, and nilestriol. Differentially expressed mRNAs were functionally annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Some histone cluster genes were validated by real-time PCR, and the effects of radioprotectant combinations were monitored by survival analysis. We found that these radioprotectants increased the induction of lncRNAs and mRNAs. miRNA, lncRNA, and mRNA expression patterns were similar with amifostine and CBLB502, but not nilestriol. The radioprotectants exhibited mostly opposite effects against IR-induced miRNAs, lncRNAs, and mRNAs while inducing a common histone gene downregulation following IR, mainly via nucleosome assembly and related signaling pathways. Notably, the effects of nilestriol significantly complemented those of amisfostine or CBLB502; low-dose drug combinations resulted in better radioprotective effects in pretreated mice. Thus, we present histone gene downregulation by radioprotectants, together with the biological functions of miRNA, lncRNA, and mRNA, to explain the mechanism underlying radioprotection.
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The nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome is involved in various acute and chronic liver diseases, however, it is not clear whether NLRP3 contributes to d-Galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced acute liver failure (ALF). This study aims to investigate the role of NLRP3 inflammasome in D-GalN/LPS-induced fatal hepatitis. We found that Nlrp3-/- and WT mice showed similar mortality against a lethal dose of D-GalN/LPS treatment. Serum ALT and AST levels, as well as liver necrosis area and hepatocyte apoptosis, were not significantly different between Nlrp3-/- and WT mice at 6 h after D-GalN/LPS injection. Moreover, the numbers of intrahepatic F4/80+ cells and Ly6G+ cells were comparable in two genotype mice following D-GalN/LPS treatment. Besides, Nlrp3-/- mice had reduced IL-1ß levels but similar TNF-α, IL-6, and MCP-1 levels compared with WT mice upon D-GalN/LPS administration. Our findings revealed that NLRP3 ablation does not protect mice from D-GalN/LPS-induced fatal hepatitis and has a marginal effect on intrahepatic inflammatory response upon D-GalN/LPS treatment. This suggests that NLRP3 inflammasome does not appear to be a major contributor to D-GalN/LPS-induced ALF.
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Fallo Hepático Agudo/etiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Galactosamina , Inflamasomas/metabolismo , Inflamasomas/fisiología , Interleucina-1beta/sangre , Lipopolisacáridos , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/inmunología , Fallo Hepático Agudo/metabolismo , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.
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Eritropoyesis/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secuencia Conservada , Células Precursoras Eritroides/citología , Técnicas de Silenciamiento del Gen , Trasplante de Células Madre Hematopoyéticas , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/química , Hígado/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Trasplante Heterólogo , Ubiquitinación , Regulación hacia ArribaRESUMEN
There is increasing evidence that the expression of non-coding RNA and mRNA (messenger RNA) is significantly altered following high-dose ionizing radiation (IR), and their expression may play a critical role in cellular responses to IR. However, the role of non-coding RNA and mRNA in radiation protection, especially in the nervous system, remains unknown. In this study, microarray profiles were used to determine microRNA (miRNA), long non-coding RNA (lncRNA), and mRNA expression in the hypothalamus of mice that were pretreated with amifostine and subsequently exposed to high-dose IR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. We found that fewer miRNAs, lncRNAs, and mRNAs were induced by amifostine pre-treatment in exposed mice, which exhibited antagonistic effects compared to IR, indicating that amifostine attenuated the IR-induced effects on RNA profiles. GO and KEGG pathway analyses showed changes in a variety of signaling pathways involved in inflammatory responses during radioprotection following amifostine pre-treatment in exposed mice. Taken together, our study revealed that amifostine treatment altered or attenuated miRNA, lncRNA, and mRNA expression in the hypothalamus of exposed mice. These data provide a resource to further elucidate the mechanisms underlying amifostine-mediated radioprotection in the hypothalamus.
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Amifostina/farmacología , Radioisótopos de Cobalto/efectos adversos , Rayos gamma/efectos adversos , Hipotálamo/efectos de la radiación , MicroARNs/efectos de la radiación , ARN Largo no Codificante/efectos de la radiación , ARN Mensajero/efectos de la radiación , Protectores contra Radiación/farmacología , Transcriptoma/efectos de la radiación , Animales , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Irradiación Corporal Total/efectos adversosRESUMEN
During human erythroid maturation, Hsp70 translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. Failure of Hsp70 to localize to the nucleus was found in Myelodysplastic syndrome (MDS) erythroblasts and can induce dyserythropoiesis, with arrest of maturation and death of erythroblasts. However, the mechanism of the nuclear trafficking of Hsp70 in erythroblasts remains unknown. Here, we found the hematopoietic transcriptional regulator, EDAG, to be a novel binding partner of Hsp70 that forms a protein complex with Hsp70 and GATA-1 during human normal erythroid differentiation. EDAG overexpression blocked the cytoplasmic translocation of Hsp70 induced by EPO deprivation, inhibited GATA-1 degradation, thereby promoting erythroid maturation in an Hsp70-dependent manner. Furthermore, in myelodysplastic syndrome (MDS) patients with dyserythropoiesis, EDAG is dramatically down-regulated, and forced expression of EDAG has been found to restore the localization of Hsp70 in the nucleus and elevate the protein level of GATA-1 to a significant extent. In addition, EDAG rescued the dyserythropoiesis of MDS patients by increasing erythroid differentiation and decreasing cell apoptosis. This study demonstrates the molecular mechanism of Hsp70 nuclear sustaining during erythroid maturation and establishes that EDAG might be a suitable therapeutic target for dyserythropoiesis in MDS patients.
Asunto(s)
Núcleo Celular/metabolismo , Eritroblastos/metabolismo , Eritropoyesis/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Síndromes Mielodisplásicos/metabolismo , Proteínas Nucleares/metabolismo , Apoptosis/fisiología , Caspasa 3/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Citoplasma/metabolismo , Regulación de la Expresión Génica/fisiología , Enfermedades Hematológicas/metabolismo , HumanosRESUMEN
During large-scale acute radiation exposure, rapidly distinguishing exposed individuals from nonexposed individuals is necessary. Identifying those exposed to high and potentially lethal radiation doses, and in need of immediate treatment, is especially important. To address this and find plasma biomarkers to assess ionizing radiation-induced mortality in the early stages, mice were administered a whole-body lethal dose of γ radiation, and radiation-induced damage was evaluated. Multiple blood biomarkers were screened using an antibody array, followed by validation using enzyme-linked immunoassay. The results revealed that irradiation (IR)-induced mortality in mice and caused body weight and blood platelet losses in deceased mice compared to surviving mice. The levels of certain proteins differed after IR between these 2 groups. Specific proteins in preirradiated mice were also found to potentiate radiosensitivity. Plasma levels of interleukin (IL)-22, urokinase, resistin, and IL-6 were associated with radiation-induced mortality in irradiated mice and may be useful as potential mortality predictors. Our results suggest that estimating the levels of certain plasma proteins is a promising alternative to conventional cytogenetic biodosimetry to accurately identify individuals exposed to high radiation doses and those at risk of death due to exposure. This strategy would facilitate the rapid triage of individuals requiring immediate and intensive medical treatment.
RESUMEN
In space, multiple unique environmental factors, particularly microgravity and space radiation, pose a constant threat to astronaut health. MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are functional RNAs that play critical roles in regulating multiple cellular processes. To gain insight into the role of non-coding RNAs in response to radiation and microgravity, we analyzed RNA expression profiles in human lymphoblastoid TK6 cells incubated for 24â¯h under static or rotating conditions to stimulate microgravity in space, after 2-Gy γ-ray irradiation. The expression of 14 lncRNAs and 17 mRNAs (differentially-expressed genes, DEGs) was found to be significantly downregulated under simulated microgravity conditions. In contrast, irradiation upregulated 55 lncRNAs and 56 DEGs, whereas only one lncRNA, but no DEGs, was downregulated. Furthermore, two miRNAs, 70 lncRNAs, and 87 DEGs showed significantly altered expression in response to simulated microgravity after irradiation, and these changes were independently induced by irradiation and simulated microgravity. GO enrichment and KEGG pathway analyses indicated that the associated target genes showed similar patterns to the noncoding RNAs and were suggested to be involved in the immune/inflammatory response including LPS/TLR, TNF, and NF-κB signaling pathways. However, synergistic effects on RNA expression and cellular responses were also observed with a combination of simulated microgravity and irradiation based on microarray and RT-PCR analysis. Together, our results indicate that simulated microgravity and irradiation additively alter expression patterns but synergistically modulate the expression levels of RNAs and their target genes in human lymphoblastoid cells.