RESUMO
Iron is mostly devoted to the hemoglobinization of erythrocytes for oxygen transport. However, emerging evidence points to a broader role for the metal in hematopoiesis, including the formation of the immune system. Iron availability in mammalian cells is controlled by iron-regulatory protein 1 (IRP1) and IRP2. We report that global disruption of both IRP1 and IRP2 in adult mice impairs neutrophil development and differentiation in the bone marrow, yielding immature neutrophils with abnormally high glycolytic and autophagic activity, resulting in neutropenia. IRPs promote neutrophil differentiation in a cell intrinsic manner by securing cellular iron supply together with transcriptional control of neutropoiesis to facilitate differentiation to fully mature neutrophils. Unlike neutrophils, monocyte count was not affected by IRP and iron deficiency, suggesting a lineage-specific effect of iron on myeloid output. This study unveils the previously unrecognized importance of IRPs and iron metabolism in the formation of a major branch of the innate immune system.
Assuntos
Medula Óssea , Neutrófilos , Animais , Medula Óssea/metabolismo , Hematopoese , Hemoglobinas/metabolismo , Homeostase , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteínas Reguladoras de Ferro/metabolismo , Mamíferos/metabolismo , Camundongos , Neutrófilos/metabolismo , Oxigênio/metabolismoRESUMO
BACKGROUND & AIMS: The insulin-like growth-factor 2 (IGF2) mRNA binding protein p62 is highly expressed in hepatocellular carcinoma tissue. Still, its potential role in liver disease is largely unknown. In this study, we investigated pathophysiological implications of p62 overexpression in mice. METHODS: We generated mice overexpressing p62 under a LAP-promotor. mRNA expression levels and stability were examined by real-time RT-PCR. Allele-specific expression of Igf2 and H19 was assessed after crossing mice with SD7 animals. The Igf2 downstream mediators pAKT and PTEN were determined by Western blot. RESULTS: Hepatic p62 overexpression neither induced inflammatory processes nor liver damage. However, 2.5week old transgenic animals displayed a steatotic phenotype and improved glucose tolerance. p62 overexpression induced the expression of the imprinted genes Igf2 and H19 and their transcriptional regulator Aire (autoimmune regulator). Neither monoallelic expression nor mRNA stability of Igf2 and H19 was affected. Investigating Igf2 downstream signalling pathways showed increased AKT activation and attenuated PTEN expression. CONCLUSIONS: The induction of a steatotic phenotype implies that p62 plays a role in hepatic pathophysiology.
Assuntos
Fator de Crescimento Insulin-Like II/genética , Fígado/patologia , Fatores de Transcrição/genética , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Expressão Gênica/fisiologia , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Humanos , Fígado/fisiologia , Camundongos , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica , Fenótipo , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Fator de Transcrição TFIIH , Fatores de Transcrição/metabolismoRESUMO
Supplemental Digital Content is available in the text.
RESUMO
The harmful effects of exposure to benzo[alpha]pyrene (B[alpha]P), which is a neurotoxic pollutant, on mammalian neurodevelopment and/or behaviour as yet remain widely unclear. In the present investigation, we evaluated the impact of the lactational exposure to B[alpha]P on postnatal development of pups and behaviour of young mice. The neurobiological effects of B[alpha]P during lactation were also evaluated on pups' brain. Here, we found that lactational exposure to B[alpha]P at 2 and 20mg/kg affects the neuromaturation of pups by significantly decreasing their reflex as highlighted in surface righting reflex and negative geotaxis tests. However, we noted a significant increase in muscular strength of lactationally B[alpha]P mg/kg-exposed pups, which was probably due to the impact of the exposure to this toxic compound on body weight gain. At the pup stage, lactational exposure to B[alpha]P also provoked a neurobiological change, which was assessed by determination of neuronal receptor gene expression. Indeed, a significant reduction in gene expression of 5HT(1A) receptors in pups exposed to B[alpha]P through lactation was found in comparison to controls. Additionally, attenuation in the expression of MOR(1) mRNA was observed, but statistically significant only in animals receiving the higher dose. Neither the expression levels of ADRA(1D) nor GABA(A) mRNA were altered. Interestingly, the harmful effects of lactational exposure to B[alpha]P on behaviour and cognitive function were still found despite a long post-weaning period. Young mice whose mothers were exposed to B[alpha]P displayed a disinhibition behaviour towards the aversive spaces of the elevated plus maze. Furthermore, a significant increase of spontaneous alternation in the Y-maze was observed, but only in young mice whose mothers were orally exposed to the lower dose of B[alpha]P. Our results suggest a close link between the neurobiological change highlighted in pups' brain and the different behavioural disturbances observed during postnatal development period until young adult stage.
Assuntos
Comportamento Animal/efeitos dos fármacos , Benzo(a)pireno/toxicidade , Encéfalo/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Exposição Materna/efeitos adversos , Animais , Animais Recém-Nascidos , Ansiedade/induzido quimicamente , Benzo(a)pireno/farmacocinética , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Poluentes Ambientais/farmacocinética , Feminino , Lactação , Masculino , Memória/efeitos dos fármacos , Camundongos , Atividade Motora/efeitos dos fármacos , Distribuição Aleatória , Receptores Adrenérgicos alfa 1/biossíntese , Receptores Adrenérgicos alfa 1/genética , Receptores de GABA-A/biossíntese , Receptores de GABA-A/genética , Receptores Opioides mu/biossíntese , Receptores Opioides mu/genética , Receptores de Serotonina/biossíntese , Receptores de Serotonina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have shown previously that during myocardial ischemia/reperfusion (MI/R), toll-like receptor 2 (TLR2) signaling regulates connexin 43 (Cx43) subcellular localization and function and dampens arrhythmia formation. We aimed to identify sites capable of TLR2-dependent redox modification within Cx43. Post-ischemic TLR2-/- or wild-type (WT) mouse hearts were analyzed by OxICAT. Cx43 was mutated to exclude redox modification and transfected into HL-1 cardiomyocytes (CM) that were challenged with a TLR2 agonist. We identified Cys260 of Cx43 to be susceptible to reversible oxidation MI/R; TLR2-/- leads to reduced H2O2 production in post-ischemic isolated mitochondria and subsequently reduced oxidation of Cx43 at Cys260. Cx43 was dephosphorylated in WT, while phosphorylation was preserved in TLR2-/-. Mutation of Cx43 (C260A) and lentiviral transfection in HL-1 CM accelerated pacemaker activity and reduced activity after TLR2 ligand stimulation. We here provide evidence for TLR2-dependent reversible oxidation of Cx43 at Cys260, which led to decreased Cx43 phosphorylation and affected CM pacemaker frequency and intercellular communication.
Assuntos
Arritmias Cardíacas/metabolismo , Conexina 43/metabolismo , Frequência Cardíaca , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Receptor 2 Toll-Like/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Comunicação Celular , Linhagem Celular , Conexina 43/deficiência , Conexina 43/genética , Cisteína , Modelos Animais de Doenças , Peróxido de Hidrogênio/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Oxirredução , Fosforilação , Transdução de Sinais , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genéticaRESUMO
Temporally controlled gene deletion provides a powerful technique for examination of gene function in vivo. To permit use of this technology in the study of cardiac pacemaking, we attempted to generate a mouse line expressing an inducible Cre recombinase selectively in cardiac pacemaker cells. The tamoxifen-inducible CreER(T2) construct was 'knocked in' into the pacemaker channel HCN4 locus. In the absence of inducing agent, recombination was undetectable in HCN4-KiT mice. After injection of tamoxifen, highly selective and efficient recombination was observed in the sinoatrial and atrioventricular node. Expression of Cre and tamoxifen per se did not affect cardiac rhythm, basal heart rate and heart rate modulation. By crossing these animals with floxed HCN4 mice, complete deletion of this gene in the sinoatrial node could be achieved. HCN4-KiT mice represent the first tool for the temporally controlled inactivation of floxed target genes selectively in the conduction system of the murine heart.
Assuntos
Antagonistas de Estrogênios/farmacologia , Deleção de Genes , Sistema de Condução Cardíaco/metabolismo , Integrases/biossíntese , Recombinação Genética/efeitos dos fármacos , Tamoxifeno/farmacologia , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Integrases/genética , Camundongos , Camundongos Transgênicos , Locos de Características Quantitativas/genética , Recombinação Genética/genéticaRESUMO
BACKGROUND: Ischemia-reperfusion edema is a common early complication after lung transplantation where the hypoxia-induced vascular endothelial growth factor (VEGF)-A plays a pivotal role. It remains unclear whether a VEGF blockade is beneficial in lung transplantation. METHODS: VEGF-A blockade was investigated in an orthotopic rat model of lung transplantation. VEGF-A antibody was added into the preservation solution alone (α-VEGF D/-), in the preservation solution and systemically to the recipient before reperfusion (α-VEGF D/R), or applied to the recipient alone before reperfusion (α-VEGF -/R). Forty-eight hours after lung transplantation, left lungs were collected and wet-to-dry ratio, Western blotting, RT-PCR, and immunohistology were performed. RESULTS: VEGF-A blockade in α-VEGF D/-, α-VEGF D/R, and α-VEGF -/R resulted in neutralization of tissue VEGF-A. Reperfusion edema was only reduced in α-VEGF D/R and α-VEGF D/- groups versus Perfadex controls. Some α-VEGF -/R rats showed a hyperinflammation leading to increased pro-inflammatory cytokine expressions as well as increased edema. Whereas generally the α-VEGF D/- group showed decreased inflammation, the combination with anti-VEGF treatment to the recipient resulted in a pro-inflammatory and a pro-apoptotic phenotype. Short-term survival, however, was not significantly different in all groups as compared to the controls. In the α-VEGF (D/R) or (D/-) groups, animals mainly died from arterial thromboembolisms and in the α-VEGF (-/R) group, hyperinflammation was the main cause of death. CONCLUSION: VEGF-A directly contributes to the formation of a reperfusion edema, which might be reduced by its blockade. However, the α-VEGF effect on the endothelial integrity might also favor arterial thrombosis formation.
Assuntos
Edema/metabolismo , Transplante de Pulmão , Traumatismo por Reperfusão/metabolismo , Tromboembolia/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose , Artérias/patologia , Modelos Animais de Doenças , Hipóxia , Inflamação , Masculino , Soluções para Preservação de Órgãos , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Trombose , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Benzo[α]pyrene (B[α]P) is a ubiquitous environmental pollutant exhibiting adverse effects on cognitive function and behaviour. In this study, depressive or antidepressive effects of B[α]P were investigated. Here, we report that a subacute B[α]P oral exposure (0.02-0.2 mg/kg) increases mobility behaviour in female adult mice in the tail suspension test, but not in the forced swimming test, without altering locomotion, suggesting that the tail suspension test was a more sensitive indicator of B[α]P-induced neurobehavioural disturbance. This might be because of differences in neurochemical substrates and pathways, mediating the performance in these behavioural models of depression. The effect of B[α]P on female adult mice in the tail suspension test was similar to that obtained with subacute treatment of the antidepressant reference drug imipramine (10 mg/kg). Therefore, B[α]P at 0.02 mg/kg and 0.2 mg/kg induces an antidepressant-like effect in mice, suggesting a neurobehavioural disturbance after oral exposure to this environmental compound. Furthermore, oral exposure to B[α]P at 0.02 mg/kg significantly increased gene expression levels of the brain receptors 5-hydroxytryptamine (serotonin) 1A (5HT(1A)) and alpha-1D adrenergic (ADRA(1D)). In summary, the presented findings suggest that subacute oral exposure to B[α]P results in behavioural changes in female adult mice, possibly caused by alterations in the serotoninergic and adrenergic systems.
Assuntos
Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Benzo(a)pireno/farmacologia , Depressão/tratamento farmacológico , Atividade Motora/efeitos dos fármacos , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Administração Oral , Fatores Etários , Animais , Antidepressivos/administração & dosagem , Benzo(a)pireno/administração & dosagem , Depressão/genética , Depressão/metabolismo , Depressão/psicologia , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/metabolismo , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Fatores SexuaisRESUMO
We aimed to determine whether 3-nitropropionic acid (3-NPA) preconditioning protects rat livers against warm ischemia/reperfusion injury. We hypothesized that 3-NPA mediates its protective effects by Bcl-2 upregulation. Brown-Norway rats (200 g) were injected with 3-NPA (10 mg/kg intraperitoneally) 24 h before 90 min of selective warm in situ ischemia. In additional experiments, 30-day survival was studied after 90 min of warm liver ischemia and resection of nonischemic liver tissue. We demonstrate increased mRNA and protein levels of Bcl-2 by real-time polymerase chain reaction, immunohistochemistry, and Western blot analysis in 3-NPA-pretreated rats. All treated animals survived, whereas all untreated rats died within 3 days after selective ischemia and resection of the nonischemic tissue. This corresponded well with a significant decrease of caspases 3 and 9 activity at 1 h of reperfusion after preconditioning with 3-NPA as compared with untreated rats. The histological sections showed protection of liver tissue after 3-NPA by reduction of apoptotic and oncotic tissue damage. Lipid peroxidation in liver tissue was reduced after 3-NPA preconditioning. We show that subtoxic doses of the mitochondrial toxin 3-NPA induces tolerance to warm liver ischemia in rats associated by synthesis of Bcl-2. Bcl-2 upregulation might protect against the postischemic burst of reactive oxygen species and therefore reduces apoptotic- and oncotic-related cell death.