RESUMEN
Ultradian oscillators are cyclically expressed genes with a period of less than 24h, found in the major signalling pathways. The Notch effector hairy and enhancer of split Hes genes are ultradian oscillators. The physiological signals that synchronise and entrain Hes oscillators remain poorly understood. We investigated whether cellular metabolism modulates Hes1 cyclic expression. We demonstrated that, in mouse myoblasts (C2C12), Hes1 oscillation depends on reactive oxygen species (ROS), which are generated by the mitochondria electron transport chain and by NADPH oxidases NOXs. In vitro, the regulation of Hes1 by ROS occurs via the calcium-mediated signalling. The modulation of Hes1 by ROS was relevant in vivo, since perturbing ROS homeostasis was sufficient to alter Medaka (Oryzias latipes) somitogenesis, a process that is dependent on Hes1 ultradian oscillation during embryo development. Moreover, in a Medaka model for human microphthalmia with linear skin lesions syndrome, in which mitochondrial ROS homeostasis was impaired, we documented important somitogenesis defects and the deregulation of Hes homologues genes involved in somitogenesis. Notably, both molecular and developmental defects were rescued by antioxidant treatments. Our studies provide the first evidence of a coupling between cellular redox metabolism and an ultradian biological oscillator with important pathophysiological implication for somitogenesis.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Relojes Biológicos/fisiología , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Oryzias/genética , Especies Reactivas de Oxígeno/metabolismo , Somitos/patología , Animales , Antioxidantes/farmacología , Calcio/metabolismo , Células Cultivadas , Complejo III de Transporte de Electrones/metabolismo , Embrión no Mamífero/metabolismo , Humanos , Hibridación in Situ , Ratones , Microftalmía/metabolismo , Microftalmía/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , NADPH Oxidasas/metabolismo , Oryzias/embriología , Transducción de Señal , Anomalías Cutáneas/metabolismo , Anomalías Cutáneas/patología , Somitos/embriología , Somitos/metabolismo , Síndrome , Factor de Transcripción HES-1RESUMEN
miRNAs are small non-coding RNAs able to modulate target gene expression. It has been postulated that miRNAs confer robustness to biological processes, but clear experimental evidence is still missing. Here, using a synthetic biological approach, we demonstrate that microRNAs provide phenotypic robustness to transcriptional regulatory networks by buffering fluctuations in protein levels. We construct a network motif in mammalian cells exhibiting a 'toggle-switch' phenotype in which two alternative protein expression levels define its ON and OFF states. The motif consists of an inducible transcription factor that self-regulates its own transcription and that of a miRNA against the transcription factor itself. We confirm, using mathematical modelling and experimental approaches, that the microRNA confers robustness to the toggle-switch by enabling the cell to maintain and transmit its state. When absent, a dramatic increase in protein noise level occurs, causing the cell to randomly switch between the two states.
Asunto(s)
Factor de Transcripción E2F1/genética , Retroalimentación Fisiológica , Redes Reguladoras de Genes , MicroARNs/genética , Modelos Genéticos , Fenotipo , Animales , Células CHO , Cricetulus , Factor de Transcripción E2F1/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos , Lentivirus/genética , MicroARNs/metabolismo , Biosíntesis de Proteínas , Procesos Estocásticos , Biología Sintética , Transcripción GenéticaRESUMEN
CD44 is a marker of cancer stem-like cells and epithelial-mesenchymal transition that is overexpressed in many cancer types, including thyroid carcinoma. At extracellular and intramembranous domains, CD44 undergoes sequential metalloprotease- and γ-secretase-mediated proteolytic cleavage, releasing the intracellular protein fragment CD44-ICD, which translocates to the nucleus and activates gene transcription. Here, we show that CD44-ICD binds to the transcription factor CREB, increasing S133 phosphorylation and CREB-mediated gene transcription. CD44-ICD enhanced CREB recruitment to the cyclin D1 promoter, promoting cyclin D1 transcription and cell proliferation. Thyroid carcinoma cells harboring activated RET/PTC, RAS, or BRAF oncogenes exhibited CD44 cleavage and CD44-ICD accumulation. Chemical blockade of RET/PTC, BRAF, metalloprotease, or γ-secretase were each sufficient to blunt CD44 processing. Furthermore, thyroid cancer cell proliferation was obstructed by RNA interference-mediated knockdown of CD44 or inhibition of γ-secretase and adoptive CD44-ICD overexpression rescued cell proliferation. Together, these findings reveal a CD44-CREB signaling pathway that is needed to sustain cancer cell proliferation, potentially offering new molecular targets for therapeutic intervention in thyroid carcinoma.