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It is not clear as to whether weight bearing and ambulation may affect bone growth. Our goal was to study the role of mechanical loading (one of the components of ambulation) on endochondral ossification and longitudinal bone growth. Thus, we applied cyclical, biologically relevant strains for a prolonged time period (4 weeks) to one tibia of juvenile mice, while using the contralateral one as an internal control. By the end of the 4-week loading period, the mean tibial growth of the loaded tibiae was significantly greater than that of the unloaded tibiae. The mean height and the mean area of the loaded tibial growth plates were greater than those of the unloaded tibiae. In addition, in female mice we found a greater expression of PTHrP in the loaded tibial growth plates than in the unloaded ones. Lastly, microCT analysis revealed no difference between loaded and unloaded tibiae with respect to the fraction of bone volume relative to the total volume of the region of interest or the tibial trabecular bone volume. Thus, our findings suggest that intermittent compressive forces applied on tibiae at mild-moderate strain magnitude induce a significant and persistent longitudinal bone growth. PTHrP expressed in the growth plate appears to be one growth factor responsible for stimulating endochondral ossification and bone growth in female mice.
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Placa de Crecimiento , Proteína Relacionada con la Hormona Paratiroidea , Tibia , Soporte de Peso , Animales , Femenino , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/genética , Tibia/metabolismo , Tibia/crecimiento & desarrollo , Tibia/diagnóstico por imagen , Placa de Crecimiento/metabolismo , Placa de Crecimiento/crecimiento & desarrollo , Ratones , Soporte de Peso/fisiología , Estrés Mecánico , Ratones Endogámicos C57BL , Desarrollo Óseo , Osteogénesis/fisiologíaRESUMEN
Many anticancer therapies cause serious cardiovascular complications that degrade quality of life and cause early mortality in treated patients. Specifically, doxorubicin is known as an effective anticancer agent that causes cardiomyopathy in treated patients. There has been growing interest in defining the role of endothelial cells in cardiac damage by doxorubicin. We have shown in the present study that endothelial nuclei accumulate more intravenously administered doxorubicin than other cardiac cell types. Doxorubicin enhanced cardiac production of the transforming growth factor-ß (TGF-ß) ligands and nuclear translocation of phospho-Smad3 in both cultured and in vivo cardiac endothelial cells. To examine the role of the TGF-ß/mothers against decapentaplegic homolog 3 (Smad3) pathway in cardiac damage by doxorubicin, we used both Smad3 shRNA stable endothelial cell lines and Smad3-knockout mice. We demonstrated using endothelial transcriptome analysis that upregulation of the TGF-ß and inflammatory cytokine/cytokine receptor pathways, as well as suppression of cell cycle and angiogenesis by doxorubicin, were alleviated in Smad3-deficient endothelial cells. The results of transcriptomic analysis were validated using qPCR, immunoblotting, and ex vivo aortic ring sprouting assays. Similarly, increased cardiac expression of cytokines and chemokines observed in treated wild-type mice was diminished in treated Smad3-knockout animals. We also detected increased end-diastolic diameter and depressed systolic function in doxorubicin-treated wild-type but not Smad3-knockout mice. This work provides evidence for the critical role of the canonical TGF-ß/Smad3 pathway in cardiac damage by doxorubicin.NEW & NOTEWORTHY Microvascular endothelial cells in the heart accumulate more intravenously administered doxorubicin than nonendothelial cardiac cell types. The treatment enhanced the TGF-ß/Smad3 pathway and elicited endothelial cell senescence and inflammatory responses followed by adverse cardiac remodeling and dysfunction in wild-type but not Smad3-deficient animals. Our study suggests that the TGF-ß/Smad3 pathway contributes to the development of doxorubicin cardiomyopathy and the potential value of novel approaches to ameliorate cardiotoxicity by targeting the Smad3 transcription factor.
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Cardiomiopatías , Células Endoteliales , Ratones , Animales , Células Endoteliales/metabolismo , Calidad de Vida , Proteína smad3/genética , Proteína smad3/metabolismo , Doxorrubicina/toxicidad , Factor de Crecimiento Transformador beta/metabolismo , Ratones NoqueadosRESUMEN
Increased fluid-flow shear stress (FFSS) contributes to hyperfiltration-induced podocyte and glomerular injury resulting in progression of chronic kidney disease (CKD). We reported that increased FFSS in vitro and in vivo upregulates PGE2 receptor EP2 (but not EP4 expression), COX2-PGE2 -EP2 axis, and EP2-linked Akt-GSK3ß-ß-catenin signaling pathway in podocytes. To understand and use the disparities between PGE2 receptors, specific agonists, and antagonists of EP2 and EP4 were used to assess phosphorylation of Akt, GSK3ß and ß-catenin in podocytes using Western blotting, glomerular filtration barrier function using in vitro albumin permeability (Palb ) assay, and mitigation of hyperfiltration-induced injury in unilaterally nephrectomized (UNX) mice at 1 and 6 months. Results show an increase in Palb by PGE2 , EP2 agonist (EP2AGO ) and EP4 antagonist (EP4ANT ), but not by EP2 antagonist (EP2ANT ) or EP4 agonist (EP4AGO ). Pretreatment with EP2ANT blocked the effect of PGE2 or EP2AGO on Palb . Modulation of EP2 and EP4 also induced opposite effects on phosphorylation of Akt and ß-Catenin. Individual agonists or antagonists of EP2 or EP4 did not induce significant improvement in albuminuria in UNX mice. However, treatment with a combination EP2ANT + EP4AGO for 1 or 6 months caused a robust decrease in albuminuria. EP2ANT + EP4AGO combination did not impact adaptive hypertrophy or increased serum creatinine. Observed differences between expression of EP2 and EP4 on the glomerular barrier highlight these receptors as potential targets for intervention. Safe and effective mitigating effect of EP2ANT + EP4AGO presents a novel opportunity to delay the progression of hyperfiltration-associated CKD as seen in transplant donors.
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Subtipo EP2 de Receptores de Prostaglandina E , Insuficiencia Renal Crónica , Albúminas , Albuminuria , Animales , Creatinina , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hormonas Esteroides Gonadales , Ratones , Proteínas Proto-Oncogénicas c-akt , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E , beta CateninaRESUMEN
Gene editing introduces stable mutations into the genome and has powerful applications extending from research to clinical gene therapy. CRISPR-Cas9 gene editing can be employed to study directly the functional impact of stable gene knockout, activation, and knockdown. Here, we describe the end-to-end methodology by which we employ genome-wide CRISPR-Cas9 knockout to study drug toxicity using acetaminophen (APAP) in a hepatocellular carcinoma liver model as an example. This methodology can be extended to other proliferative cell types and chemical metabolic and toxicity models. By employing a massively parallelized genome-wide knockout model, the genes responsible for cellular toxicity and proliferation may be assayed concurrently. Resultant data are interrogated in the context of existing gene expression data, pathway analysis, drug-gene interactions, and orthogonal confirmatory assays to better understand the metabolic mechanisms.
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Acetaminofén , Sistemas CRISPR-Cas , Acetaminofén/efectos adversos , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Genoma , HepatocitosRESUMEN
Phenotypic variations of chromosome 22q11.2 deletion syndrome (22qDS) have unclear explanations. T cell lymphopenia in 22qDS related to varying degrees of thymic hypoplasia contributes to the phenotypic heterogeneity. No phenotype correlation with genotype or deletion size is known for lymphopenia. We investigated gene expression in human T cells of participants with and without 22qDS and T cells of participants with 22qDS with low or normal T cells. Peripheral blood was collected from participants aged 5-8 y. Immune function was checked. RNA sequencing was completed on isolated T cells, and differential gene expression profiles of T cells between 22qDS and healthy control subjects were established. A total of 360 genes were differentially expressed (q < 0.05) between T cells of patients with 22qDS (n = 13) and healthy control subjects (n = 6) (log2 fold change range, -2.0747, 15.6724). We compared gene expression between participants with 22qDS with low (n = 7) and normal T cell counts (n = 6), finding 94 genes that were differentially expressed (q < 0.05) (log2 fold change range, -4.5445, 5.1297). Twenty-nine genes correlated with T cell counts and markers CD3, CD4, CD8, and CD45RA+CD4 (R ≥ 0.8). We found significantly differentially expressed genes in participants with 22qDS compared with healthy control subjects and in participants with 22qDS with low T cell counts compared with those with normal T cell counts. Several enriched pathways suggest a role of T cells in defective communication between T cells and the innate immune system in 22qDS. Among these, the liver X receptor/retinoid X receptor pathway was noted to show several differentially expressed genes affecting participants with 22qDS compared with healthy control subjects and more so those with low T cell counts than in those with normal T cell counts.
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Síndrome de DiGeorge , Linfopenia , Cromosomas , Síndrome de DiGeorge/genética , Humanos , Receptores X del Hígado/genética , Linfopenia/genética , Receptores X Retinoide/genética , Linfocitos T , TranscriptomaRESUMEN
Manipulation of the microbiome is a rational treatment strategy for inflammatory bowel disease (IBD). Compared to the colon and terminal ileum (TI), understanding of the microbial composition in the duodenum is sparse. This gap in knowledge is especially significant for children with Crohn's disease (CD) because the prevalence of duodenal CD is higher in children than in adults. Our aim was to characterize the bacterial composition of the mucosally-adherent duodenal microbiome in children with and without CD as a first step toward development of targeted IBD treatment strategies at this disease location. Fresh-frozen mucosal biopsies were obtained from the duodenum and TI of children with treatment-naïve CD and age- and sex-matched controls. Extracted DNA was analyzed for sequence variation in the 16S ribosomal RNA bacterial gene region V4 (Novogene; Beijing, China). Bacterial relative abundance, alpha and beta composition, and diversity, were compared across duodenal and TI samples from the controls and CD groups with and without chronic active inflammation (118 samples from 73 children total; approx. 50% CD), using UniFrac dissimilarity coefficients (α < 0.05), Linear Discriminant Analysis Effect Size (LEfSe) analysis (LDA score ≥ 2), and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis. The relationships between bacterial abundance, sex, age, concomitant medication use, and villous length were assessed. The microbial composition in the duodenum was significantly different from the TI in the control population(R-value = 0.558, p = 0.001) and in children with active CD (R-value = 0.301, p = 0.001). Significant differences in bacterial abundance were noted between the control and CD duodena (LDA > 4). The duodenum of children without CD was characterized by increased abundance in Pseudomonodales, whereas the actively inflamed duodenum in CD was characterized by increased abundance of Bacteroidales, specifically the family Prevotellaceae. This trend is opposite of previously published observations of microbial composition in the TI, where active inflammation was associated with a relative decrease in the abundance of Bacteroidetes and an increase in Proteobacteria, including Pseudomonadales. No statistically significant correlations were noted between abundance and age, sex, concomitant medication use or villous length, except for Bacteroidetes, which significantly decreased in abundance in the TI with age (p = 0.048). The pediatric duodenal microbiome is distinct from the TI and characterized by an increased abundance of Pseudomonodales and Spirochetes in healthy children, and an increased abundance of Bacteroidales in active CD patients.
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BACKGROUND: Escherichia coli is a major neonatal pathogen and the leading cause of early-onset sepsis in preterm newborns. Maternal E. coli strains are transmitted to the newborn causing invasive neonatal disease. However, there is a lack of data regarding the phenotypic and genotypic characterization of E. coli strains colonizing pregnant women during labor. METHODS: This prospective study performed at the University of Oklahoma Medical Center (OUHSC) from March 2014 to December 2015, aimed to investigate the colonization rate, and the phylogeny, antibiotic resistance traits, and invasive properties of E. coli strains colonizing the cervix of fifty pregnant women diagnosed with preterm labor (PTL). Molecular analyses including bacterial whole-genome sequencing (WGS), were performed to examine phylogenetic relationships among the colonizing strains and compare them with WGS data of representative invasive neonatal E. coli isolates. Phenotypic and genotypic antibiotic resistance traits were investigated. The bacteria's ability to invade epithelial cells in vitro was determined. RESULTS: We recruited fifty women in PTL. Cervical samples yielded E. coli in 12 % (n=6). The mean gestational age was 32.5 (SD±3.19) weeks. None delivered an infant with E. coli disease. Phenotypic and genotypic antibiotic resistance testing did not overall demonstrate extensive drug resistance traits among the cervical E. coli isolates, however, one isolate was multi-drug resistant. The isolates belonged to five different phylogroups, and WGS analyses assigned each to individual multi-locus sequence types. Single nucleotide polymorphism-based comparisons of cervical E. coli strains with six representative neonatal E. coli bacteremia isolates demonstrated that only half of the cervical E. coli isolates were phylogenetically related to these neonatal invasive strains. Moreover, WGS comparisons showed that each cervical E. coli isolate had distinct genomic regions that were not shared with neonatal E. coli isolates. Cervical and neonatal E. coli isolates that were most closely related at the phylogenetic level had similar invasion capacity into intestinal epithelial cells. In contrast, phylogenetically dissimilar cervical E. coli strains were the least invasive among all isolates. CONCLUSIONS: This pilot study showed that a minority of women in PTL were colonized in the cervix with E. coli, and colonizing strains were not phylogenetically uniformly representative of E. coli strains that commonly cause invasive disease in newborns. Larger studies are needed to determine the molecular characteristics of E. coli strains colonizing pregnant women associated with an increased risk of neonatal septicemia.
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Cuello del Útero/microbiología , Escherichia coli/aislamiento & purificación , Trabajo de Parto Prematuro/microbiología , Adulto , Antibacterianos/farmacología , Línea Celular , Farmacorresistencia Bacteriana/genética , Células Epiteliales/microbiología , Escherichia coli/clasificación , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Femenino , Genoma Bacteriano/genética , Humanos , Recién Nacido , Pruebas de Sensibilidad Microbiana , Sepsis Neonatal/microbiología , Filogenia , Proyectos Piloto , Embarazo , Estudios Prospectivos , Adulto JovenRESUMEN
Systemic inflammation in pregnant obese women is associated with 1.5- to 2-fold increase in serum Interleukin-6 (IL-6) and newborns with lower kidney/body weight ratio but the role of IL-6 in increased susceptibility to chronic kidney (CKD) in adult progeny is not known. Since IL-6 crosses the placental barrier, we administered recombinant IL-6 (10 pg/g) to pregnant mice starting at mid-gestation yielded newborns with lower body (p < 0.001) and kidney (p < 0.001) weights. Histomorphometry indicated decreased nephrogenic zone width (p = 0.039) with increased numbers of mature glomeruli (p = 0.002) and pre-tubular aggregates (p = 0.041). Accelerated maturation in IL-6 newborns was suggested by early expression of podocyte-specific protein podocin in glomeruli, increased 5-methyl-cytosine (LC-MS analysis for CpG DNA methylation) and altered expression of certain genes of cell-cycle and apoptosis (RT-qPCR array-analysis). Western blotting showed upregulated pJAK2/pSTAT3. Thus, treating dams with IL-6 as a surrogate provides newborns to study effects of maternal systemic inflammation on future susceptibility to CKD in adulthood.
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Interleucina-6/efectos adversos , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Apoptosis/efectos de los fármacos , Peso al Nacer/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Femenino , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/patologíaRESUMEN
Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function in vivo. FFSS-treated cultured podocytes show upregulated AKT-GSK3ß-ß-catenin signaling. The present study was undertaken to confirm (i) the activation of ß-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the ß-galactosidase reporter gene for ß-catenin activation, (ii) ß-catenin translocation in FFSS-treated mouse podocytes, and (iii) ß-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased ß-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy (p < 0.001 at 4 weeks; p = 0.16 at 12 weeks) and X-gal staining (p = 0.008 at 4 weeks; p = 0.65 at 12 weeks). Immunofluorescence microscopy showed a significant increase in phospho-ß-catenin (Ser552, p = 0.005) at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-ß-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-ß-catenin (Ser552) but not phospho-ß-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified ß-catenin as a key upstream regulator. We conclude that transcription factor ß-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.
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Tasa de Filtración Glomerular , Mecanotransducción Celular , Podocitos/metabolismo , Riñón Único/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Genes fos , Operón Lac , Factor de Unión 1 al Potenciador Linfoide/genética , Ratones Transgénicos , Podocitos/patología , Regiones Promotoras Genéticas , Riñón Único/genética , Riñón Único/patología , Riñón Único/fisiopatología , Estrés Mecánico , Factor de Transcripción 3/genética , beta Catenina/genéticaRESUMEN
The molecular mechanisms by which endothelial cells (ECs) regulate pulmonary vascularization and contribute to alveolar epithelial cell development during lung morphogenesis remain unknown. We tested the hypothesis that delta-like 4 (DLL4), an EC Notch ligand, is critical for alveolarization by combining lung mapping and functional studies in human tissue and DLL4-haploinsufficient mice (Dll4+/lacz). DLL4 expressed in a PECAM-restricted manner in capillaries, arteries, and the alveolar septum from the canalicular to alveolar stage in mice and humans. Dll4 haploinsufficiency resulted in exuberant, nondirectional vascular patterning at E17.5 and P6, followed by smaller capillaries and fewer intermediate blood vessels at P14. Vascular defects coincided with polarization of lung EC expression toward JAG1-NICD-HES1 signature and decreased tip cell-like (Car4) markers. Dll4+/lacZ mice had impaired terminal bronchiole development at the canalicular stage and impaired alveolarization upon lung maturity. We discovered that alveolar type I cell (Aqp5) markers progressively decreased in Dll4+/lacZ mice after birth. Moreover, in human lung EC, DLL4 deficiency programmed a hypersprouting angiogenic phenotype cell autonomously. In conclusion, DLL4 is expressed from the canalicular to alveolar stage in mice and humans, and Dll4 haploinsufficiency programs dysmorphic microvascularization, impairing alveolarization. Our study reveals an obligate role for DLL4-regulated angiogenesis in distal lung morphogenesis.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al Calcio/metabolismo , Pulmón/irrigación sanguínea , Pulmón/embriología , Proteínas Adaptadoras Transductoras de Señales/genética , Células Epiteliales Alveolares/fisiología , Animales , Proteínas de Unión al Calcio/genética , Regulación del Desarrollo de la Expresión Génica , Haploinsuficiencia , Humanos , Hipoxia , Ratones Endogámicos C57BL , Ratones Mutantes , Neovascularización Fisiológica/genética , Alveolos Pulmonares/citología , Alveolos Pulmonares/embriología , Alveolos Pulmonares/metabolismoRESUMEN
Systemic sepsis is a known risk factor for bronchopulmonary dysplasia (BPD) in premature infants, a disease characterized by dysregulated angiogenesis and impaired vascular and alveolar development. We have previoulsy reported that systemic endotoxin dysregulates pulmonary angiogenesis resulting in alveolar simplification mimicking BPD in neonatal mice, but the underlying mechanisms remain unclear. We undertook an unbiased discovery approach to identify novel signaling pathways programming sepsis-induced deviant lung angiogenesis. Pulmonary endothelial cells (EC) were isolated for RNA-Seq from newborn C57BL/6 mice treated with intraperitoneal lipopolysaccharide (LPS) to mimic systemic sepsis. LPS significantly differentially-regulated 269 genes after 6 h, and 1,934 genes after 24 h. Using bioinformatics, we linked 6 h genes previously unknown to be modulated by LPS to 24 h genes known to regulate angiogenesis/vasculogenesis to identify pathways programming deviant angiogenesis. An immortalized primary human lung EC (HPMEC-im) line was generated by SV40 transduction to facilitate mechanistic studies. RT-PCR and transcription factor binding analysis identified FOSL1 (FOS like 1) as a transcriptional regulator of LPS-induced downstream angiogenic or vasculogenic genes. Over-expression and silencing studies of FOSL1 in immortalized and primary HPMEC demonstrated that baseline and LPS-induced expression of ADAM8, CXCR2, HPX, LRG1, PROK2, and RNF213 was regulated by FOSL1. FOSL1 silencing impaired LPS-induced in vitro HPMEC angiogenesis. In conclusion, we identified FOSL1 as a novel regulator of sepsis-induced deviant angiogenic signaling in mouse lung EC and human fetal HPMEC.
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Displasia Broncopulmonar , Lipopolisacáridos/toxicidad , Pulmón , Neovascularización Patológica , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Displasia Broncopulmonar/inducido químicamente , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Ratones , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patologíaRESUMEN
The ultrafiltrate flow over the major processes and cell body generates fluid flow shear stress (FFSS) on podocytes. Hyperfiltration-associated increase in FFSS can lead to podocyte injury and detachment. Previously, we showed that FFSS-induced upregulation of the cyclooxygenase 2 (COX2)-PGE2-prostaglandin E receptor 2 (EP2) axis in podocytes activates Akt-glycogen synthase kinase-3ß-ß-catenin and MAPK/ERK signaling in response to FFSS. Integrative MultiOmics Pathway Resolution (IMPRes) is a new bioinformatic tool that enables simultaneous time-series analysis of more than two groups to identify pathways and molecular connections. In the present study, we used previously characterized COX2 [prostaglandin-endoperoxide synthase 2 (Ptgs2)], EP2 (Ptger2), and ß1-catenin (Ctnnb1) as "seed genes" from an array data set of four groups analyzed over a time course. The 3 seed genes shared 7 pathways and 50 genes of 14 pathways and 89 genes identified by IMPRes. A composite of signaling pathways highlighted the temporal molecular connections during mechanotransduction signaling in FFSS-treated podocytes. We investigated the "proteoglycans in cancer" and "galactose metabolism" pathways predicted by IMPRes. A custom-designed PCR array validated 60.7% of the genes predicted by IMPRes analysis, including genes for the above-named pathways. Further validation using Western blot analysis showed increased expression of phosho-Erbb2, phospho-mammalian target of rapamycin (mTOR), CD44, and hexokinase II (Hk2); decreased total Erbb2, galactose mutarotase (Galm), and ß-1,4-galactosyltransferase 1 (B4galt1); and unchanged total mTOR and AKT3. These findings corroborate our previously reported results. This study demonstrates the potential of the IMPRes method to identify novel pathways. Identifying the "proteoglycans in cancer" and "galactose metabolism" pathways has generated a lead to study the significance of FFSS-induced glycocalyx remodeling and possible detachment of podocytes from the glomerular matrix.
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Podocitos/metabolismo , Proteoglicanos/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Estrés Mecánico , Activación Transcripcional/fisiología , Ciclooxigenasa 2/metabolismo , Glomérulos Renales/metabolismo , Mecanotransducción Celular/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Congenital chloride diarrhea (CCD) in a newborn is a rare autosomal recessive disorder with life-threatening complications, requiring early diagnostics and treatment to prevent severe dehydration and infant mortality. SLC26A3 rs386833481 (c.392C>G; p.P131R) gene polymorphism is an important genetic determinant of CCD. Here, we report the influence of the non-synonymous SLC26A3 variant rs386833481 gene polymorphism on the function of the epithelial barrier and the potential mechanisms of these effects. RESULTS: We found that P131R-SLC26A3 increased dysfunction of the epithelial barrier compared with wild type SLC26A3 in human colonic Caco-2 and mouse colonic CMT-93 cells. When P131R-SLC26A3 was subsequently reverted to wild type, the epithelial barrier function was restored similar to wild type cells. Further study demonstrated that variant P131R-SLC26A3 disrupts function of epithelial barrier through two distinct molecular mechanisms: (a) decreasing SLC26A3 expression through a ubiquitination pathway and (b) disrupting a key interaction with its partner ZO-1/CFTR, thereby increasing the epithelial permeability. CONCLUSION: Our study provides an important insight of SLC26A3 SNPs in the regulation of the epithelial permeability and indicates that SLC26A3 rs386833481 is likely a causative mutation in the dysfunction of epithelial barrier of CCD, and correction of this SNP or increasing SLC26A3 function could be therapeutically beneficial for chronic diarrhea diseases.
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Nicotinamide phosphoribosyltransferase (NAMPT) functions in NAD synthesis, apoptosis, and inflammation. Dysregulation of NAMPT has been associated with several inflammatory diseases, including rheumatoid arthritis (RA). The purpose of this study was to investigate NAMPT's role in arthritis using mouse and cellular models. Collagen-induced arthritis (CIA) in DBA/1J Nampt +/- mice was evaluated by ELISA, micro-CT, and RNA-sequencing (RNA-seq). In vitro Nampt loss-of-function and gain-of-function studies on osteoclastogenesis were examined by TRAP staining, nascent RNA capture, luciferase reporter assays, and ChIP-PCR. Nampt-deficient mice presented with suppressed inflammatory bone destruction and disease progression in a CIA mouse model. Nampt expression was required for the epigenetic regulation of the Nfatc1 promoter and osteoclastogenesis. Finally, RNA-seq identified 690 differentially expressed genes in whole ankle joints which associated (P < 0.05) with Nampt expression and CIA. Selected target was validated by RT-PCR or functional characterization. We have provided evidence that NAMPT functions as a genetic risk factor and a potential therapeutic target to RA.
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Acetaminophen (APAP) is a commonly used analgesic responsible for more than half of acute liver failure cases. Identification of previously unknown genetic risk factors would provide mechanistic insights and novel therapeutic targets for APAP-induced liver injury. This study used a genome-wide CRISPR-Cas9 screen to evaluate genes that are protective against, or cause susceptibility to, APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR-Cas9 gene knockouts were treated with 15 mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap of expression data from APAP overdose studies, and 3) predicted affected biological pathways. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. This study illustrated the power of a genome-wide CRISPR-Cas9 screen to systematically identify novel genes involved in APAP-induced hepatotoxicity and to provide potential targets to develop novel therapeutic modalities.
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Acetaminofén/efectos adversos , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Genes Reguladores , Hepatocitos/metabolismo , Animales , Línea Celular Tumoral , Bases de Datos como Asunto , Regulación de la Expresión Génica , Células HEK293 , Hepatocitos/efectos de la radiación , Humanos , Masculino , Ratones Endogámicos C57BL , Reproducibilidad de los Resultados , Transducción de Señal/genéticaRESUMEN
Acetaminophen is commonly used to reduce pain and fever. Unfortunately, overdose of acetaminophen is a leading cause of acute liver injury and failure in many developed countries. The majority of acetaminophen is safely metabolized in the liver and excreted in the urine; however, a small percentage is converted to the highly reactive N-acetyl-p-benzoquinone imine (NAPQI). At therapeutic doses, NAPQI is inactivated by glutathione S-transferases, but at toxic levels, excess NAPQI forms reactive protein adducts that lead to hepatotoxicity. Individual variability in the response to both therapeutic and toxic levels of acetaminophen suggests a genetic component is involved in acetaminophen metabolism. In this review, we evaluate the genetic association studies that have identified 147 single nucleotide polymorphisms linked to acetaminophen-induced hepatotoxicity. The identification of novel genetic markers for acetaminophen-induced hepatotoxicity provides a rich resource for further evaluation and may lead to improved prognosis, prevention, and treatment.
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Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Hígado/efectos de los fármacos , Polimorfismo de Nucleótido Simple/genética , Animales , Benzoquinonas/farmacología , Glutatión/metabolismo , Humanos , Iminas/farmacología , Hígado/metabolismoRESUMEN
Acetaminophen overdose is the most common cause of acute liver injury (ALI) or acute liver failure in the United States. Its pathogenetic mechanisms are incompletely understood. Additional studies are warranted to identify new genetic risk factors for more mechanistic insights and new therapeutic target discoveries. The objective of this study was to explore the role and mechanisms of nicotinamide phosphoribosyltransferase (NAMPT) in acetaminophen-induced ALI. C57BL/6 Nampt gene wild-type (Nampt+/+), heterozygous knockout (Nampt+/-), and overexpression (NamptOE) mice were treated with overdose of acetaminophen, followed by histologic, biochemical, and transcriptomic evaluation of liver injury. The mechanism of Nampt in acetaminophen-induced hepatocytic toxicity was also explored in cultured primary hepatocytes. Three lines of evidence have convergently demonstrated that acetaminophen overdose triggers the most severe oxidative stress and necrosis, and the highest expression of key necrosis driving genes in Nampt+/- mice, whereas the effects in NamptOE mice were least severe relative to Nampt+/+ mice. Treatment of P7C3-A20, a small chemical molecule up-regulator of Nampt, ameliorated acetaminophen-induced mouse ALI over the reagent control. These findings support the fact that NAMPT protects against acetaminophen-induced ALI.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Citocinas/fisiología , Nicotinamida Fosforribosiltransferasa/fisiología , Sustancias Protectoras , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés OxidativoRESUMEN
Although a deficiency of surfactant protein B (SFTPB) has been associated with lung injury, SFTPB expression has not yet been linked with nicotinamide phosphoribosyltransferase (NAMPT), a potential biomarker of acute lung injury (ALI). The effects of Nampt in the pulmonary epithelial cell on both SFTPB expression and lung inflammation were investigated in a LPS-induced ALI mouse model. Pulmonary epithelial cell-specific knockdown of Nampt gene expression, achieved by the crossing of Nampt gene exon 2 floxed mice with mice expressing epithelial-specific transgene Cre or by the use of epithelial-specific expression of anti-Nampt antibody cDNA, significantly attenuated LPS-induced ALI. Knockdown of Nampt expression was accompanied by lower levels of bronchoalveolar lavage (BAL) neutrophil infiltrates, total protein and TNF-α levels, as well as lower lung injury scores. Notably, Nampt knockdown was also associated with significantly increased BAL SFTPB levels relative to the wild-type control mice. Down-regulation of NAMPT increased the expression of SFTPB and rescued TNF-α-induced inhibition of SFTPB, whereas overexpression of NAMPT inhibited SFTPB expression in both H441 and A549 cells. Inhibition of NAMPT up-regulated SFTPB expression by enhancing histone acetylation to increase its transcription. Additional data indicated that these effects were mainly mediated by NAMPT nonenzymatic function via the JNK pathway. This study shows that pulmonary epithelial cell-specific knockdown of NAMPT expression attenuated ALI, in part, via up-regulation of SFTPB expression. Thus, epithelial cell-specific knockdown of Nampt may be a potential new and viable therapeutic modality to ALI.-Bi, G., Wu, L., Huang, P., Islam, S., Heruth, D. P., Zhang, L. Q., Li, D.-Y., Sampath, V., Huang, W., Simon, B. A., Easley, R. B., Ye, S. Q. Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Células Epiteliales Alveolares/metabolismo , Citocinas/genética , Nicotinamida Fosforribosiltransferasa/genética , Surfactantes Pulmonares/metabolismo , Lesión Pulmonar Aguda/genética , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Histonas/metabolismo , Humanos , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , Nicotinamida Fosforribosiltransferasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Lower plasma nicotinamide phosphoribosyltransferase (NAMPT) levels are associated with improved response to methotrexate (MTX) in patients with juvenile idiopathic arthritis. Cell-based studies confirmed that reduced cellular NAMPT activity potentiates the pharmacologic activity of MTX; however, the mechanism of this interaction has yet to be defined. Therefore, in this study, we investigate the mechanism of enhanced pharmacologic activity of MTX in NAMPT-deficient A549 cells. Small interfering RNA-based silencing of NAMPT expression resulted in a greater than 3-fold increase in sensitivity to MTX (P < 0.005) that was completely reversed by supplementation with folinic acid. Despite a 68% reduction in cellular NAD levels in NAMPT-deficient cells, no change in expression or activity of dihydrofolate reductase was observed and uptake of MTX was not significantly altered. MTX did not potentiate the depletion of cellular NAD levels, but NAMPT-deficient cells had significant elevations in levels of intermediates of de novo purine biosynthesis and were 4-fold more sensitive to depletion of ATP by MTX (P < 0.005). Supplementation with hypoxanthine and thymidine completely reversed the antiproliferative activity of MTX in NAMPT-deficient cells and corresponded to repletion of the cellular ATP pool without any effect on NAD levels. Together, these findings demonstrate that increased MTX activity with decreased NAMPT expression is dependent on the antifolate activity of MTX and is driven by enhanced sensitivity to the ATP-depleting effects of MTX. For the first time, these findings provide mechanistic details to explain the increase in pharmacological activity of MTX under conditions of reduced NAMPT activity.