RESUMEN
Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.
Asunto(s)
Modelos Animales de Enfermedad , Terapia Genética/métodos , Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno/terapia , ARN Mensajero/genética , Animales , Línea Celular Tumoral , Citocinas/sangre , Citocinas/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno/genética , Enfermedad del Almacenamiento de Glucógeno/patología , Células HeLa , Humanos , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nanopartículas/administración & dosificación , Nanopartículas/química , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Resultado del Tratamiento , Triglicéridos/metabolismoRESUMEN
Epigenetic, transcriptional and signaling processes in the nucleolus regulate rRNA transcription and cell growth. We report here that the tumor suppressor ING1b binds rDNA, regulates rDNA chromatin modifications and affects nucleolar localization of mTOR to modulate rRNA levels. ING1 represses rDNA transcription by recruiting HDAC1 to rDNA loci, increasing its association with the NoRC complex and deacetylating the histone H3K9 and H3K27 marks of active transcription. Loss of ING1 enhances nucleolar localization of phospho-mTOR and its association with Raptor and GßL, even during rapamycin treatment. ING1 inhibits rDNA transcription by inhibiting UBF activity and its interaction with mTOR. Regulation of rDNA heterochromatin and rRNA synthesis by ING1 is also apparent during normal cell growth and during cell stress. Moreover, this function was also important during PMA induced differentiation of THP1 cells, since knocking down ING1 affected the process by inhibiting rRNA transcriptional repression. These observations show that ING1 regulates the nucleolar epigenome and rDNA transcription suggesting that regulation of protein synthesis might serve as the basis for ING1 function as a type II tumor suppressor.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Diferenciación Celular/genética , Ensamble y Desensamble de Cromatina , Epigénesis Genética , Silenciador del Gen , Genes Supresores de Tumor , Glucosa/metabolismo , Histona Desacetilasa 1/metabolismo , Humanos , Proteína Inhibidora del Crecimiento 1 , Monocitos/citología , Monocitos/metabolismo , Complejos Multiproteicos/metabolismo , Unión Proteica , Transporte de Proteínas , Precursores del ARN/genética , Precursores del ARN/metabolismoRESUMEN
Cell senescence contributes to organismal aging and is induced by telomere erosion and an ensuing DNA damage signal as cells reach the end of their replicative lifespan in vitro or in vivo. Stresses induced by oncogene or tumor suppressor hyperactivation, oxidative stress, ionizing radiation and other DNA damaging agents result in forms of stress induced premature senescence (SIPS) that show similarities to replicative senescence. Since replicative senescence and SIPS occur over many days and many population doublings of the mass cultures of primary cells used to study senescence, the sequence of events that occur downstream of senescence signaling can be challenging to define. Here we compare a new model of ING1a-induced senescence with several other forms of senescence. The ING1a epigenetic regulator synchronously induces senescence in mass cultures several-fold faster than all other agents, taking 24 and 36 hours to activate the Rb/ p16INK4a, but not the p53 tumor suppressor axis to efficiently induce senescence. ING1a induces expression of intersectin 2, a scaffold protein necessary for endocytosis, altering the stoichiometry of endocytosis proteins, subsequently blocking growth factor uptake leading to activation of Rb signaling to block cell growth. ING1a acts as a novel link in the activation of the Rb pathway that can impose senescence in the absence of activating p53-mediated DNA damage signaling, and should prove useful in defining the molecular events contributing to Rb-induced senescence.
Asunto(s)
Envejecimiento Prematuro/genética , Senescencia Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Proteína de Retinoblastoma/metabolismo , Estrés Fisiológico/genética , Proteínas Supresoras de Tumor/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Línea Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/genética , Endocitosis/fisiología , Células Endoteliales/metabolismo , Humanos , Proteína Inhibidora del Crecimiento 1 , Queratinocitos/metabolismo , Homeostasis del Telómero/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Expression of the alternatively spliced ING1a tumor suppressor increases >10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated ß-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced >25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57(KIP2) cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1a-induced senescence signaling. These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype.