RESUMO
Pancreatic beta cell death is a hallmark of type 1 (T1D) and type 2 (T2D) diabetes, but the molecular mechanisms underlying this aspect of diabetic pathology are poorly understood. Here we report that expression of the microRNA (miR)-200 family is strongly induced in islets of diabetic mice and that beta cell-specific overexpression of miR-200 in mice is sufficient to induce beta cell apoptosis and lethal T2D. Conversely, mir-200 ablation in mice reduces beta cell apoptosis and ameliorates T2D. We show that miR-200 negatively regulates a conserved anti-apoptotic and stress-resistance network that includes the essential beta cell chaperone Dnajc3 (also known as p58IPK) and the caspase inhibitor Xiap. We also observed that mir-200 dosage positively controls activation of the tumor suppressor Trp53 and thereby creates a pro-apoptotic gene-expression signature found in islets of diabetic mice. Consequently, miR-200-induced T2D is suppressed by interfering with the signaling of Trp53 and Bax, a proapoptotic member of the B cell lymphoma 2 protein family. Our results reveal a crucial role for the miR-200 family in beta cell survival and the pathophysiology of diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/metabolismo , MicroRNAs/genética , Animais , Apoptose/genética , Sobrevivência Celular/genética , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP40/biossíntese , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/metabolismo , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossínteseRESUMO
The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/fisiologia , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Componente 3 do Complexo de Manutenção de Minicromossomo , Componente 4 do Complexo de Manutenção de Minicromossomo , Componente 6 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipoprotein B and factor VII) that achieved efficient (approximately 80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA.