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1.
Nat Genet ; 53(8): 1233-1242, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34326545

RESUMO

The agouti viable yellow (Avy) allele is an insertional mutation in the mouse genome caused by a variably methylated intracisternal A particle (VM-IAP) retrotransposon. Avy expressivity is sensitive to a range of early-life chemical exposures and nutritional interventions, suggesting that environmental perturbations can have long-lasting effects on the methylome. However, the extent to which VM-IAP elements are environmentally labile with phenotypic implications is unknown. Using a recently identified repertoire of VM-IAPs, we assessed the epigenetic effects of different environmental contexts. A longitudinal aging analysis indicated that VM-IAPs are stable across the murine lifespan, with only small increases in DNA methylation detected for a subset of loci. No significant effects were observed after maternal exposure to the endocrine disruptor bisphenol A, an obesogenic diet or methyl donor supplementation. A genetic mouse model of abnormal folate metabolism exhibited shifted VM-IAP methylation levels and altered VM-IAP-associated gene expression, yet these effects are likely largely driven by differential targeting by polymorphic KRAB zinc finger proteins. We conclude that epigenetic variability at retrotransposons is not predictive of environmental susceptibility.


Assuntos
Metilação de DNA , Disruptores Endócrinos/toxicidade , Obesidade/genética , Retroelementos , Animais , Compostos Benzidrílicos/toxicidade , Metilação de DNA/efeitos dos fármacos , Dieta/efeitos adversos , Epigênese Genética , Feminino , Ferredoxina-NADP Redutase/genética , Ácido Fólico/genética , Ácido Fólico/metabolismo , Deficiência de Ácido Fólico/genética , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Obesidade/etiologia , Fenóis/toxicidade , Gravidez , Efeitos Tardios da Exposição Pré-Natal
2.
Am J Physiol Lung Cell Mol Physiol ; 321(3): L555-L565, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34261337

RESUMO

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.


Assuntos
Elastase de Leucócito/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Fagocitose , Fagossomos/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Humanos , Elastase de Leucócito/genética , Lisossomos/genética , Lisossomos/patologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Mutantes , Fagossomos/genética , Fagossomos/patologia , Células RAW 264.7
3.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298981

RESUMO

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors expressed in the skin. Three PPAR isotypes, α (NRC1C1), ß or δ (NRC1C2) and γ (NRC1C3), have been identified. After activation through ligand binding, PPARs heterodimerize with the 9-cis-retinoic acid receptor (RXR), another nuclear hormone receptor, to bind to specific PPAR-responsive elements in regulatory regions of target genes mainly involved in organogenesis, cell proliferation, cell differentiation, inflammation and metabolism of lipids or carbohydrates. Endogenous PPAR ligands are fatty acids and fatty acid metabolites. In past years, much emphasis has been given to PPARα and γ in skin diseases. PPARß/δ is the least studied PPAR family member in the skin despite its key role in several important pathways regulating inflammation, keratinocyte proliferation and differentiation, metabolism and the oxidative stress response. This review focuses on the role of PPARß/δ in keratinocytes and its involvement in psoriasis and atopic dermatitis. Moreover, the relevance of targeting PPARß/δ to alleviate skin inflammation is discussed.


Assuntos
Dermatite Atópica/metabolismo , Queratinócitos/metabolismo , PPAR delta/fisiologia , Psoríase/metabolismo , Pele/metabolismo , Anaerobiose , Animais , Dimerização , Eicosanoides/metabolismo , Ácidos Graxos/metabolismo , Glicólise , Humanos , Camundongos , Camundongos Mutantes , Especificidade de Órgãos , Fosforilação , Isoformas de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Proteólise , Receptores X de Retinoides/metabolismo , Pele/patologia
4.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201747

RESUMO

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.


Assuntos
Toxinas Bacterianas/farmacologia , Encéfalo/efeitos dos fármacos , Proteínas de Escherichia coli/farmacologia , Proteína 2 de Ligação a Metil-CpG/genética , Mitocôndrias/efeitos dos fármacos , Síndrome de Rett/tratamento farmacológico , Animais , Toxinas Bacterianas/administração & dosagem , Encéfalo/metabolismo , Modelos Animais de Doenças , Proteínas de Escherichia coli/administração & dosagem , Medo/efeitos dos fármacos , Feminino , Infusões Intraventriculares , Mutação com Perda de Função , Masculino , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Camundongos Mutantes , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Síndrome de Rett/etiologia , Serina-Treonina Quinases TOR/metabolismo
5.
Am J Physiol Lung Cell Mol Physiol ; 321(2): L291-L307, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34132118

RESUMO

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Células Epiteliais Alveolares , Autofagia , Regulação da Expressão Gênica , Lesão Pulmonar , Mutação de Sentido Incorreto , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Substituição de Aminoácidos , Animais , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Camundongos , Camundongos Mutantes , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Síndrome do Desconforto Respiratório do Recém-Nascido/patologia
6.
Mol Cell ; 81(15): 3187-3204.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34157307

RESUMO

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.


Assuntos
Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Endopeptidases/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Poliubiquitina/metabolismo , Adulto , Animais , Endopeptidases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Fator 2 de Diferenciação de Crescimento/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Mutantes , Mutação , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Telangiectasia Hemorrágica Hereditária , Ubiquitina-Proteína Ligases/metabolismo
7.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071003

RESUMO

Superoxide dismutase (SOD) is a major antioxidant enzyme for superoxide removal, and cytoplasmic SOD (SOD1) is expressed as a predominant isoform in all cells. We previously reported that renal SOD1 deficiency accelerates the progression of diabetic nephropathy (DN) via increasing renal oxidative stress. To evaluate whether the degree of SOD1 expression determines regeneration capacity and sarcopenic phenotypes of skeletal muscles under incipient and advanced DN conditions, we investigated the alterations of SOD1 expression, oxidative stress marker, inflammation, fibrosis, and regeneration capacity in cardiotoxin (CTX)-injured tibialis anterior (TA) muscles of two Akita diabetic mouse models with different susceptibility to DN, DN-resistant C57BL/6-Ins2Akita and DN-prone KK/Ta-Ins2Akita mice. Here, we report that KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, exhibit delayed muscle regeneration after CTX injection, as demonstrated by the finding indicating significantly smaller average cross-sectional areas of regenerating TA muscle myofibers relative to KK/Ta-wild-type mice. Furthermore, we observed markedly reduced SOD1 expression in CTX-injected TA muscles of KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, along with increased inflammatory cell infiltration, prominent fibrosis and superoxide overproduction. Our study provides the first evidence that SOD1 reduction and the following superoxide overproduction delay skeletal muscle regeneration through induction of overt inflammation and fibrosis in a mouse model of progressive DN.


Assuntos
Nefropatias Diabéticas/complicações , Músculo Esquelético/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Sarcopenia/etiologia , Superóxido Dismutase-1/efeitos dos fármacos , Animais , Cardiotoxinas/toxicidade , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Progressão da Doença , Indução Enzimática/efeitos dos fármacos , Fibrose , Regulação Enzimológica da Expressão Gênica , Predisposição Genética para Doença , Mesângio Glomerular/patologia , Inflamação , Insulina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase-1/biossíntese , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/fisiologia , Superóxidos/metabolismo
8.
Methods Mol Biol ; 2308: 235-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34057727

RESUMO

Over the last 20 years, significant progress has been made in the development of immunodeficient mouse models that now represents the gold standard tool in stem cell biology research. The latest major improvement has been the use of biomaterials in these xenogeneic mouse models to generate human "bone marrow like" tissues, which not only provides a more relevant xenograft model but can also potentially enable us to delineate the interactions that are specific between human bone marrow cells. There are a number of biomaterials and strategies to create humanized niches in immunodeficient mouse models, and the methods can also differ significantly among various research institutes. Here, we describe a protocol to create a humanized 3D collagen-based scaffold human niche in immunodeficient mouse model(s). This humanized in vivo model provides a powerful technique for understanding the human BM microenvironment and the role it plays in the regulation of normal as well as malignant hematopoiesis.


Assuntos
Hematopoese , Transplante de Células-Tronco Hematopoéticas/instrumentação , Células-Tronco Hematopoéticas/fisiologia , Hospedeiro Imunocomprometido , Nicho de Células-Tronco , Tecidos Suporte , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Mutantes , Fenótipo , Transplante Heterólogo
9.
Am J Respir Cell Mol Biol ; 65(3): 272-287, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33938785

RESUMO

Right ventricular (RV) function is the predominant determinant of survival in patients with pulmonary arterial hypertension (PAH). In preclinical models, pharmacological activation of BMP (bone morphogenetic protein) signaling with FK506 (tacrolimus) improved RV function by decreasing RV afterload. FK506 therapy further stabilized three patients with end-stage PAH. Whether FK506 has direct effects on the pressure-overloaded right ventricle is yet unknown. We hypothesized that increasing cardiac BMP signaling with FK506 improves RV structure and function in a model of fixed RV afterload after pulmonary artery banding (PAB). Direct cardiac effects of FK506 on the microvasculature and RV fibrosis were studied after surgical PAB in wild-type and heterozygous Bmpr2 mutant mice. RV function and strain were assessed longitudinally via cardiac magnetic resonance imaging during continuous FK506 infusion. Genetic lineage tracing of endothelial cells (ECs) was performed to assess the contribution of ECs to fibrosis. Molecular mechanistic studies were performed in human cardiac fibroblasts and ECs. In mice, low BMP signaling in the right ventricle exaggerated PAB-induced RV fibrosis. FK506 therapy restored cardiac BMP signaling, reduced RV fibrosis in a BMP-dependent manner independent from its immunosuppressive effect, preserved RV capillarization, and improved RV function and strain over the time course of disease. Endothelial mesenchymal transition was a rare event and did not significantly contribute to cardiac fibrosis after PAB. Mechanistically, FK506 required ALK1 in human cardiac fibroblasts as a BMPR2 co-receptor to reduce TGFß1-induced proliferation and collagen production. Our study demonstrates that increasing cardiac BMP signaling with FK506 improves RV structure and function independent from its previously described beneficial effects on pulmonary vascular remodeling.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/farmacologia , Função Ventricular Direita/efeitos dos fármacos , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Proteínas Morfogenéticas Ósseas/genética , Fibroblastos/metabolismo , Fibrose , Humanos , Masculino , Camundongos , Camundongos Mutantes , Miocárdio/metabolismo , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/genética , Transdução de Sinais/genética , Função Ventricular Direita/genética
10.
Development ; 148(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33913480

RESUMO

Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenoteratozoospermia. We have identified protease serine 50 (PRSS50) as having a crucial role in sperm development, because Prss50-null mice presented with impaired fertility and sperm tail abnormalities. PRSS50 could also be involved in centrosome function because these mice showed a threefold increase in acephalic sperm (head-tail junction defect), sperm with multiple heads (spermatid division defect) and sperm with multiple tails, including novel two conjoined sperm (complete or partial parts of several flagellum on the same plasma membrane). Our data support that, in the testis, as in tumorigenesis, PRSS50 activates NFκB target genes, such as the centromere protein leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1), which is required for heterochromatin maintenance. Prss50-null testes have increased IκκB, and reduced LRWD1 and histone expression. Low levels of de-repressed histone markers, such as H3K9me3, in the Prss50-null mouse testis may cause increases in post-meiosis proteins, such as AKAP4, affecting sperm formation. We provide important insights into the complex mechanisms of sperm development, the importance of testis proteases in fertility and a novel mechanism for MMAF.


Assuntos
Fertilidade , Serina Proteases/metabolismo , Cauda do Espermatozoide/enzimologia , Testículo/enzimologia , Animais , Astenozoospermia/enzimologia , Astenozoospermia/genética , Heterocromatina/enzimologia , Heterocromatina/genética , Histonas/biossíntese , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Serina Proteases/deficiência , Cabeça do Espermatozoide/enzimologia
11.
Biochem Biophys Res Commun ; 558: 126-133, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33915326

RESUMO

Myocardial ischemia/reperfusion (I/R) injury is a major determinant of morbidity and mortality in patients undergoing treatment for cardiac disease. A variety of treatments are reported to have benefits against reperfusion injury, yet their cardioprotective effects seem to be diminished in obesity, and the underlying mechanism remains elusive. In this study, we found that db/db mice exhibit cardiac hyper-O-GlcNAcylation. In parallel, palmitate treatment (200 mM; 12 h) in H9c2 cells showed an increase in global protein O-GlcNAcylation, along with an impaired insulin response against reperfusion injury. To investigate whether O-GlcNAcylation underlies this phenomenon, glucosamine was used to increase global protein O-GlcNAc levels. Interestingly, histological staining, electrophysiological studies, serum cardiac markers and oxidative stress biomarker assays showed that preischemic treatment with glucosamine attenuated insulin cardioprotection against myocardial infarction, arrhythmia and oxidative stress. Mechanistically, glucosamine treatment decreased insulin-stimulated Akt phosphorylation, a key modulator of cell survival. Furthermore, inhibition of O-GlcNAcylation via 6-diazo-5-oxo-l-norleucine (DON) apparently increased insulin-induced Akt phosphorylation and restored its cardioprotective response against reperfusion injury in palmitate-induced insulin-resistant H9c2 cells. Our findings demonstrated that obesity-induced hyper-O-GlcNAcylation might contribute to the attenuation of insulin cardioprotection against I/R injury.


Assuntos
Acetilglucosamina/metabolismo , Arritmias Cardíacas/complicações , Arritmias Cardíacas/metabolismo , Insulina/metabolismo , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Animais , Cardiotônicos/metabolismo , Hipóxia Celular , Linhagem Celular , Diazo-Oxo-Norleucina/farmacologia , Modelos Animais de Doenças , Glicosilação/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos
12.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805516

RESUMO

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O2•-) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O2•- in mice. However, the pathological contribution of XO-derived O2•- production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O2•- derived from XOR in Sod1-/- mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1-/- mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1-/- mice. These results showed that XOR-mediated O2•- production is relatively uninvolved in the age-related pathologies in Sod1-/- mice.


Assuntos
Envelhecimento/fisiologia , Superóxido Dismutase-1/genética , Superóxidos/metabolismo , Xantina Desidrogenase/metabolismo , Acetofenonas/farmacologia , Envelhecimento/efeitos dos fármacos , Alopurinol/farmacologia , Anemia/genética , Animais , Fígado Gorduroso/genética , Camundongos Mutantes , Atrofia Muscular/genética , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Superóxido Dismutase-1/metabolismo , Xantina Desidrogenase/antagonistas & inibidores , Xantina Desidrogenase/genética
13.
Biochem Biophys Res Commun ; 555: 128-133, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33813271

RESUMO

mRNA decapping is a critical step in posttranscriptional regulation of gene expression in eukaryotes. Although Dcp1a is a well characterized and widely conserved mRNA decapping factor, little is known about its physiological function. To extend our understanding of Dcp1a function in vivo, we employed a transgenic rescue strategy to produce Dcp1a-deficient mice using the CRISPR/Cas9 system. This approach arrowed us to generate heterozygous Dcp1a mice and define the phenotype of Dcp1a-deficient embryos. We found that expression of Dcp1a protein, which is detectable in most mouse tissues, was developmentally regulated through embryonic growth, and that depletion of the Dcp1a gene resulted in embryonic lethality around embryonic day 10.5 (E10.5) concomitant with massive growth retardation and cardiac developmental defects. Moreover, the embryonic lethality was fully rescued by transgenic expression of exogenous human Dcp1a. Together, our results suggest that Dcp1a is required for embryonic growth.


Assuntos
Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transativadores/genética , Transativadores/metabolismo , Animais , Sistemas CRISPR-Cas , Feminino , Coração/embriologia , Cardiopatias Congênitas/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos
14.
Biochem Biophys Res Commun ; 555: 74-80, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33813279

RESUMO

The engagement of the receptor for advanced glycation end-products (receptor for AGEs, RAGE) with diverse ligands could elicit chronic vascular inflammation, such as atherosclerosis. Binding of cytoplasmic tail RAGE (ctRAGE) to diaphanous-related formin 1 (Diaph1) is known to yield RAGE intracellular signal transduction and subsequent cellular responses. However, the effectiveness of an inhibitor of the ctRAGE/Diaph1 interaction in attenuating the development of atherosclerosis is unclear. In this study, using macrophages from Ager+/+ and Ager-/- mice, we validated the effects of an inhibitor on AGEs-RAGE-induced foam cell formation. The inhibitor significantly suppressed AGEs-RAGE-evoked Rac1 activity, cell invasion, and uptake of oxidized low-density lipoprotein, as well as AGEs-induced NF-κB activation and upregulation of proinflammatory gene expression. Moreover, expression of Il-10, an anti-inflammatory gene, was restored by this antagonist. These findings suggest that the RAGE-Diaph1 inhibitor could be a potential therapeutic drug against RAGE-related diseases, such as chronic inflammation and atherosclerosis.


Assuntos
Células Espumosas/metabolismo , Macrófagos Peritoneais/patologia , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Expressão Gênica , Inflamação/genética , Inflamação/patologia , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Neuropeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Receptor para Produtos Finais de Glicação Avançada/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismo
15.
Biochem Biophys Res Commun ; 555: 168-174, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33819747

RESUMO

When animals are infected with helminthic parasites, resistant hosts mount type II helper T (Th2) immune responses to expel worms. Recent studies have clearly shown that epithelial cell-derived cytokines contribute to the induction of Th2 immune responses. Here we demonstrate the role of endogenous thymic stromal lymphopoietin (TSLP) for protection against Strongyloides venezuelensis (S. venezuelensis) infection, utilizing TSLP receptor-deficient Crlf2-/- mice. The number of eggs per gram of feces (EPG) and worm burden were significantly higher in Crlf2-/- mice than in wild type (WT) mice. S. venezuelensis infection induced Tslp mRNA expression in the skin, lung, and intestine and also facilitated the accumulation of mast cells in the intestine in a TSLP-dependent manner. Furthermore, CD4+ T cells from S. venezuelensis-infected Crlf2-/- mice showed diminished capacity to produce Th2 cytokines in the early stage of infection. Finally, CD4+ cell-depleted Crlf2-/- mice still showed higher EPG counts and worm burden than CD4+ cell-depleted WT mice, indicating that TSLP contributes to protecting mice against S. venezuelensis infection in both CD4+ T cell-dependent and -independent manners.


Assuntos
Linfócitos T CD4-Positivos/parasitologia , Citocinas/fisiologia , Estrongiloidíase/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Resistência à Doença/fisiologia , Fezes/parasitologia , Interações Hospedeiro-Parasita , Imunoglobulina E/sangue , Imunoglobulinas/genética , Intestinos/parasitologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Citocinas/genética , Estrongiloidíase/parasitologia
16.
Mol Cell Endocrinol ; 529: 111263, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33811970

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19). The main organ affected in this infection is the lung and the virus uses the angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the target cells. In this context, a controversy raised regarding the use of renin-angiotensin system (RAAS) blockers, as these drugs might increase ACE2 expression in some tissues and potentially increase the risk for SARS-CoV-2 infection. This is specially concerning in diabetic patients as diabetes is a risk factor for COVID-19. METHODS: 12-week old diabetic mice (db/db) were treated with ramipril, or vehicle control for 8 weeks. Non-diabetic db/m mice were included as controls. ACE2 expression and activity were studied in lung, kidney and heart of these animals. RESULTS: Kidney ACE2 activity was increased in the db/db mice as compared to the db/m (143.2% ± 23% vs 100% ± 22.3%, p = 0.004), whereas ramipril had no significant effect. In the lung, no differences were found in ACE2 when comparing db/db mice to db/m and ramipril also had no significant effect. In the heart, diabetes decreased ACE2 activity (83% ± 16.8%, vs 100% ± 23.1% p = 0.02), and ramipril increased ACE2 significantly (83% ± 16.8% vs 98.2% ± 15%, p = 0.04). CONCLUSIONS: In a mouse model of type 2 diabetes, ramipril had no significant effect on ACE2 activity in either kidneys or in the lungs. Therefore, it is unlikely that RAAS blockers or at least angiotensin-converting enzyme inhibitors increase the risk of SARS-CoV-2 infection through increasing ACE2.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Rim/metabolismo , Pulmão/metabolismo , Miocárdio/metabolismo , Ramipril/farmacologia , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/enzimologia , COVID-19/genética , COVID-19/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Rim/patologia , Rim/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Mutantes , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , SARS-CoV-2/genética
17.
Nat Commun ; 12(1): 2046, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824347

RESUMO

Bone formation represents a heritable trait regulated by many signals and complex mechanisms. Its abnormalities manifest themselves in various diseases, including sclerosing bone disorder (SBD). Exploration of genes that cause SBD has significantly improved our understanding of the mechanisms that regulate bone formation. Here, we discover a previously unknown type of SBD in four independent families caused by bi-allelic loss-of-function pathogenic variants in TMEM53, which encodes a nuclear envelope transmembrane protein. Tmem53-/- mice recapitulate the human skeletal phenotypes. Analyses of the molecular pathophysiology using the primary cells from the Tmem53-/- mice and the TMEM53 knock-out cell lines indicates that TMEM53 inhibits BMP signaling in osteoblast lineage cells by blocking cytoplasm-nucleus translocation of BMP2-activated Smad proteins. Pathogenic variants in the patients impair the TMEM53-mediated blocking effect, thus leading to overactivated BMP signaling that promotes bone formation and contributes to the SBD phenotype. Our results establish a previously unreported SBD entity (craniotubular dysplasia, Ikegawa type) and contribute to a better understanding of the regulation of BMP signaling and bone formation.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/patologia , Proteínas de Membrana/metabolismo , Esclerose/patologia , Transdução de Sinais , Proteínas Smad/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Mutantes , Mutação/genética , Osteoblastos/patologia , Linhagem , Fosforilação , Crânio/patologia , Adulto Jovem
18.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807989

RESUMO

For a better translation from treatment designs of schizophrenia to clinical efficiency, there is a crucial need to refine preclinical animal models. In order to consider the multifactorial nature of the disorder, a new mouse model associating three factors (genetic susceptibility-partial deletion of the MAP6 gene, early-life stress-maternal separation, and pharmacological treatment-chronic Δ-9-tetrahydrocannabinol during adolescence) has recently been described. While this model depicts a schizophrenia-like phenotype, the neurobiological correlates remain unknown. Synaptic transmission and functional plasticity of the CA1 hippocampal region of male and female 3-hit mice were therefore investigated using electrophysiological recordings on the hippocampus slice. While basal excitatory transmission remained unaffected, NMDA receptor (NMDAr)-mediated long-term potentiation (LTP) triggered by theta-burst (TBS) but not by high-frequency (HFS) stimulation was impaired in 3-hit mice. Isolated NMDAr activation was not affected or even increased in female 3-hit mice, revealing a sexual dimorphism. Considering that the regulation of LTP is more prone to inhibitory tone if triggered by TBS than by HFS, the weaker potentiation in 3-hit mice suggests a deficiency of intrinsic GABA regulatory mechanisms. Indeed, NMDAr activation was increased by GABAA receptor blockade in wild-type but not in 3-hit mice. This electrophysiological study highlights dysregulations of functional properties and plasticity in hippocampal networks of 3-hit mice, one of the mechanisms suspected to contribute to the pathophysiology of schizophrenia. It also shows differences between males and females, supporting the sexual dimorphism observed in the disorder. Combined with the previously reported study, the present data reinforce the face validity of the 3-hit model that will help to consider new therapeutic strategies for psychosis.


Assuntos
Região CA1 Hipocampal/fisiopatologia , Potenciação de Longa Duração , Esquizofrenia/fisiopatologia , Transmissão Sináptica , Ritmo Teta , Animais , Região CA1 Hipocampal/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Mutantes , Esquizofrenia/genética , Esquizofrenia/patologia
19.
J Immunol ; 206(7): 1505-1514, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33658297

RESUMO

IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous Ikzf1 mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in IKZF1 are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through N-ethyl-N-nitrosourea mutagenesis, we have discovered a novel Ikzf1 mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation (Ikzf1L132P ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1L132P overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1L132P Furthermore, Ikzf1+/L132P mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of Hsf1 expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in Ikzf1L132P/L132P mice.


Assuntos
Linfócitos B/imunologia , Imunodeficiência de Variável Comum/genética , Fator de Transcrição Ikaros/genética , Mutação Puntual/genética , Animais , Formação de Anticorpos , Células HEK293 , Haploinsuficiência , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Fator de Transcrição Ikaros/metabolismo , Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células NIH 3T3
20.
J Nutr ; 151(4): 800-809, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33693772

RESUMO

BACKGROUND: While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic inheritance, and lifetime number of stem cell divisions in a tissue. Folate deficiency is generally associated with increased risk for colorectal cancer (CRC) and acute lymphocytic leukemia (ALL). Conversely, high folic acid (FA) intake has also been associated with higher CRC risk. OBJECTIVE: Our objective was to compare the effect of folate intake on mutant frequency (MF) and types of mutations in the colon and bone marrow of mice. METHODS: Five-week-old MutaMouse male mice were fed a deficient (0 mg FA/kg), control (2 mg FA/kg), or supplemented (8 mg FA/kg) diet for 20 wk. Tissue MF was assessed using the lacZ mutant assay and comparisons made by 2-factor ANOVA. LacZ mutant plaques were sequenced using next-generation sequencing, and diet-specific mutation profiles within each tissue were compared by Fisher's exact test. RESULTS: In the colon, the MF was 1.5-fold and 1.3-fold higher in mice fed the supplemented diet compared with mice fed the control (P = 0.001) and deficient (P = 0.008) diets, respectively. This contrasted with the bone marrow MF in the same mice where the MF was 1.7-fold and 1.6-fold higher in mice fed the deficient diet compared with mice fed the control (P = 0.02) and supplemented (P = 0.03) diets, respectively. Mutation profiles and signatures (mutation context) were tissue-specific. CONCLUSIONS: Our data indicate that dietary folate intake affects mutagenesis in a tissue- and dose-specific manner in mice. Mutation profiles were generally tissue- but not dose-specific, suggesting that altered cellular folate status appears to interact with endogenous mutagenic mechanisms in each tissue to create a permissive context in which specific mutation types accumulate. These data illuminate potential mechanisms underpinning differences in observed associations between folate intake/status and cancer.


Assuntos
Ácido Fólico/administração & dosagem , Taxa de Mutação , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Relação Dose-Resposta a Droga , Ácido Fólico/efeitos adversos , Ácido Fólico/sangue , Deficiência de Ácido Fólico/sangue , Deficiência de Ácido Fólico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Óperon Lac/efeitos dos fármacos , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese , Especificidade de Órgãos
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