RESUMEN
FGFR signaling is critical to development and disease pathogenesis, initiating phosphorylation-driven signaling cascades, notably the RAS-RAF-MEK-ERK and PI3 K-AKT cascades. PTEN antagonizes FGFR signaling by reducing AKT and ERK activation. Mouse lenses lacking FGFR2 exhibit microphakia and reduced ERK and AKT phosphorylation, widespread apoptosis, and defective lens fiber cell differentiation. In contrast, simultaneous deletion of both Fgfr2 and Pten restores ERK and AKT activation levels as well as lens size, cell survival and aspects of fiber cell differentiation; however, the molecular basis of this "rescue" remains undefined. We performed transcriptomic analysis by RNA sequencing of mouse lenses with conditional deletion of Fgfr2, Pten or both Fgfr2 and Pten, which reveal new molecular mechanisms that uncover how FGFR2 and PTEN signaling interact during development. The FGFR2-deficient lens transcriptome demonstrates overall loss of fiber cell identity with deregulated expression of 1448 genes. We find that ~ 60% of deregulated genes return to normal expression levels in lenses lacking both Fgfr2 and Pten. Further, application of customized filtering parameters to these RNA-seq data sets identified 68 high-priority candidate genes. Bioinformatics analyses showed that the cis-binding motif of a high-priority homeodomain transcription factor, NKX6-1, was present in the putative promoters of ~ 78% of these candidates. Finally, biochemical reporter assays demonstrate that NKX6-1 activated the expression of the high-priority candidate Rasgrp1, a RAS-activating protein. Together, these data define a novel regulatory module in which NKX6-1 directly activates Rasgrp1 expression to restore the balance of ERK and AKT activation, thus providing new insights into alternate regulation of FGFR downstream events.
Asunto(s)
Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Homeodominio/metabolismo , Microftalmía/prevención & control , Fosfohidrolasa PTEN/deficiencia , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/deficiencia , Transcriptoma , Animales , Diferenciación Celular , Proliferación Celular , Factores de Intercambio de Guanina Nucleótido/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Microftalmía/etiología , Microftalmía/patología , Fosforilación , Transducción de SeñalRESUMEN
BACKGROUND: We examined a large number of variables to generate new hypotheses regarding a wider range of risk factors for anophthalmia/microphthalmia using data mining. METHODS: Data were from the National Birth Defects Prevention Study, a multicentre, case-control study from 10 centres in the United States. There were 134 cases of "isolated" and 87 "nonisolated" (with other major birth defects) of anophthalmia/microphthalmia and 11 052 nonmalformed controls with delivery dates October 1997-December 2011. Using random forest, a data mining procedure, we compared the two case types with controls for 201 variables. Variables considered important ranked by random forest were included in a multivariable logistic regression model to estimate odds ratios and 95% confidence intervals. RESULTS: Predictors for isolated cases included paternal race/ethnicity, maternal intake of certain nutrients and foods, and childhood health problems in relatives. Using regression, inverse associations were observed with greater maternal education and with increasing intake of folate and potatoes. Odds were slightly higher with greater paternal education, for increased intake of carbohydrates and beans, and if relatives had a childhood health problem. For nonisolated cases, predictors included paternal race/ethnicity, maternal intake of certain nutrients, and smoking in the home the month before conception. Odds were higher for Hispanic fathers and smoking in the home and NSAID use the month before conception. CONCLUSIONS: Results appear to support previously hypothesised risk factors, socio-economic status, NSAID use, and inadequate folate intake, and potentially provide new areas such as passive smoking pre-pregnancy, and paternal education and ethnicity, to explore for further understanding of anophthalmia/microphthalmia.
Asunto(s)
Anoftalmos/epidemiología , Anoftalmos/etiología , Minería de Datos , Microftalmía/epidemiología , Microftalmía/etiología , Adulto , Anoftalmos/prevención & control , Antiinflamatorios no Esteroideos , Estudios de Casos y Controles , Escolaridad , Etnicidad , Femenino , Encuestas Epidemiológicas , Humanos , Recién Nacido , Masculino , Exposición Materna/efectos adversos , Exposición Materna/estadística & datos numéricos , Fenómenos Fisiologicos Nutricionales Maternos , Microftalmía/prevención & control , Oportunidad Relativa , Atención Preconceptiva/estadística & datos numéricos , Embarazo , Factores de Riesgo , Contaminación por Humo de Tabaco/efectos adversos , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND: We previously explored associations between nutrients including folate and other macro and micronutrients and risks of anophthalmia or microphthalmia in the National Birth Defects Prevention Study. In the current study, we expand those previous results with larger sample sizes and conduct analyses with an additional diet quality index using more recent data. METHODS: The National Birth Defects Prevention Study is a population-based, multicenter case-control study of over 30 major birth defects, with estimated due dates from October 1997 to December 2011. Cases were 224 infants diagnosed with anophthalmia or microphthalmia. Controls were 11,109 live-born, nonmalformed infants randomly selected by each study center. Mothers completed a standardized, computer-assisted telephone interview between 6 weeks and 24 months after delivery. Mothers responded to a shortened food frequency questionnaire, assessing their nutrient intake for the year before pregnancy, and questions about periconceptional (2 months before to 2 months after conception) vitamin supplement use. Nutrient intake quartiles were based on the intake among controls. RESULTS: Among vitamin supplement users, odds of anophthalmia/microphthalmia were decreased for women with intake levels in the highest quartile of folate (0.56, 95% confidence interval [CI] 0.32-0.98), magnesium (0.42, 95% CI 0.22-0.82), and vitamin E (0.50, 95% CI 0.29-0.89). Among women not reporting vitamin supplement use, the odds were significantly increased for beta-carotene (2.5, 95% CI 1.10-5.68) and decreased for retinol (0.37, 95% CI 0.19-0.73). CONCLUSIONS: In this expanded analysis, we observed associations for a few nutrients, specifically forms of vitamin A. However, the heterogeneity of results by form and vitamin use necessitates further inquiry.
Asunto(s)
Anoftalmos/etiología , Microftalmía/etiología , Nutrientes/uso terapéutico , Adulto , Anoftalmos/prevención & control , Estudios de Casos y Controles , Dieta , Suplementos Dietéticos , Femenino , Humanos , Recién Nacido , Entrevistas como Asunto , Microftalmía/prevención & control , Nutrientes/efectos adversos , Embarazo , Factores de Riesgo , Encuestas y CuestionariosRESUMEN
PURPOSE: To study the molecular mechanisms underlying alcohol-induced ocular anomalies in Xenopus embryos. METHODS: Xenopus embryos were exposed to various concentrations (0.1%-0.5%) of alcohol, and the subsequent effects in eye development and in eye marker gene expression were determined. To investigate the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in fetal alcohol syndrome (FAS)-associated ocular injury, two antioxidant enzymes, catalase and peroxiredoxin 5, were overexpressed in the two blastomeres of the two-cell stage Xenopus embryos. RESULTS: Exposure of Xenopus embryos to alcohol during eye development produced marked gross ocular anomalies, including microphthalmia, incomplete closure of the choroid fissure, and malformation of the retina in 40% of the eyes examined. In parallel, alcohol (0.1%-0.5%) dose dependently and significantly reduced the expression of several eye marker genes, of which TBX5, VAX2, and Pax6 were the most vulnerable. Overexpression of catalase and of cytosolic and mitochondrial peroxiredoxin 5 restored the expression of these alcohol-sensitive eye markers and significantly decreased the frequency of ocular malformation from 39% to 21%, 19%, and 13% respectively. All these enzymes reduced alcohol-induced ROS production, but only peroxiredoxin 5 inhibited RNS formation in the alcohol-treated embryos. CONCLUSIONS: The results suggest that oxidative and nitrosative stresses both contribute to alcohol-induced fetal ocular injury.
Asunto(s)
Anomalías Inducidas por Medicamentos/prevención & control , Catalasa/fisiología , Etanol/toxicidad , Anomalías del Ojo/prevención & control , Peroxidasas/fisiología , Proteínas de Xenopus , Xenopus laevis/embriología , Anomalías Inducidas por Medicamentos/etiología , Anomalías Inducidas por Medicamentos/metabolismo , Animales , Biomarcadores/análisis , Western Blotting , Coroides/anomalías , Relación Dosis-Respuesta a Droga , Embrión no Mamífero/efectos de los fármacos , Anomalías del Ojo/inducido químicamente , Anomalías del Ojo/metabolismo , Proteínas del Ojo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Microftalmía/inducido químicamente , Microftalmía/metabolismo , Microftalmía/prevención & control , Estrés Oxidativo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Peroxirredoxinas , Especies de Nitrógeno Reactivo/antagonistas & inhibidores , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras , Retina/anomalías , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismoRESUMEN
BACKGROUND: Treatment of pregnant mice with 2-chloro-2'-deoxyadenosine (2CdA) on Day 8 of gestation induces microphthalmia through a mechanism linked to the p53 tumor suppressor pathway. The present study defines the response of Day 8 mouse embryos through time with respect to pharmacologic intervention with PK11195, a ligand of the mitochondrial peripheral benzodiazepine receptor (Bzrp). METHODS: Pregnant CD-1 mice dosed with 2CdA with or without PK11195 on gestation Day 8 provided fetuses for teratologic evaluation on Day 14 and Day 17; HPLC measured pyridine nucleotides (NADH/NAD+) at 1.5 hr, RT-PCR measured mitochondrial 16S rRNA abundance at 3.0 hr, and p53 protein induction was assessed with immunostaining at 4.5 hr postexposure. RESULTS: The mean incidences of malformed fetuses were significantly higher in the 7.5 mg/kg 2CdA treatment group (50.2% malformed) vs. the 2CdA + 4.0 mg/kg PK11195 co-treatment group (4.4% malformed). Malformed fetuses displayed a range of ocular defects that included microphthalmia and keratolenticular dysgenesis (Peters anomaly). No malformations were observed in the control or PK11195 alone groups. PK11195 also protected litters from increased resorption rates and fetal weight reduction. It did not rescue early effects on NADH balance (1.5 hr) or 16S rRNA expression (3.0 hr); however, the p53 response (4.5 hr) was downgraded in 2CdA + PK11195 embryos vs. 2CdA alone. By delaying the administration of PK11195 in 1.5 hr intervals it was determined that the window for protection closed between 4.5 to 6.0 hr after 2CdA. CONCLUSIONS: The capacity of PK11195 to suppress the pathogenesis of microphthalmia implies a critical role for mitochondrial peripheral benzodiazepine receptors in the p53-dependent mode of action of 2CdA on ocular development.
Asunto(s)
Didesoxiadenosina/análogos & derivados , Didesoxiadenosina/toxicidad , Anomalías del Ojo/inducido químicamente , Isoquinolinas/uso terapéutico , Teratógenos/toxicidad , Animales , Didesoxiadenosina/administración & dosificación , Didesoxiadenosina/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Anomalías del Ojo/prevención & control , Femenino , Proteínas Fetales/biosíntesis , Proteínas Fetales/genética , Reabsorción del Feto/inducido químicamente , Reabsorción del Feto/prevención & control , Feto/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes p53 , Edad Gestacional , Isoquinolinas/farmacología , Ratones , Microftalmía/inducido químicamente , Microftalmía/prevención & control , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NAD/metabolismo , Embarazo , ARN Ribosómico 16S/biosíntesis , Receptores de GABA-A/efectos de los fármacos , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
The antiteratogenic effects of PSK, a biological response modifier, were examined using histological and developmental analysis. The whole bodies of pregnant mice were irradiated with X-rays and injected with PSK within ten minutes after irradiation on day 7 of gestation (E7). The foetuses on E18 were examined and a high incidence of malformations were observed in X-ray irradiated embryos. Microphthalmia was the most frequent malformation. PSK administration suppressed the X-ray irradiation-induced ocular anomalies in not only the frequency, as deduced by external observation, but also in histopathological changes in the retina, lens, and cornea. In particular, the incidence of lens aplasia was significantly decreased by PSK administration. Developmental analysis using E10 and E13 embryos revealed that the decrease in the incidence of histopathological changes was first observed within 72 hours after PSK administration. In addition, X-ray irradiation-induced early foetal death (E10-13) was also suppressed by PSK administration. The possible mechanisms of the antiteratogenic effects of PSK are discussed.