RESUMO
Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Diferenciação Celular/genética , Diabetes Mellitus/genética , Proteínas do Tecido Nervoso/biossíntese , Fator de Transcrição STAT3/genética , Autoimunidade/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sistemas CRISPR-Cas , Linhagem Celular , Diabetes Mellitus/etiologia , Diabetes Mellitus/patologia , Regulação da Expressão Gênica no Desenvolvimento , Glucagon/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Mutação , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Fator de Transcrição STAT3/biossínteseRESUMO
AIM: To test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain. MATERIALS AND METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes. RESULTS: In U373 cells, A-769662 (100 µM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types. CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 µM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.
