RESUMO
Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.
Assuntos
Autorrenovação Celular , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1 de Ligação ao Facilitador Linfoide/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Animais , Benzamidas/farmacologia , Autorrenovação Celular/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Edição de Genes , Fator 1-alfa Nuclear de Hepatócito/deficiência , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Ligante Coestimulador de Linfócitos T Induzíveis/antagonistas & inibidores , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/deficiência , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina/deficiência , beta Catenina/genética , Proteína AIRERESUMO
The transcription factor Stat3 is required for proliferation and pluripotency of embryonic stem cells; we have prepared and characterized fluorescent Stat3-reporter zebrafish based on repeats of minimal responsive elements. These transgenic lines mimic in vivo Stat3 expression patterns and are responsive to exogenous Stat3; notably, fluorescence is inhibited by both stat3 knockout and IL6/Jak/STAT inhibitors. At larval stages, Stat3 reporter activity correlates with proliferating regions of the brain, haematopoietic tissue and intestine. In the adult gut, the reporter is active in sparse proliferating cells, located at the base of intestinal folds, expressing the stemness marker sox9b and having the morphology of mammalian crypt base columnar cells; noteworthy, zebrafish stat3 mutants show defects in intestinal folding. Stat3 reporter activity in the gut is abolished with mutation of T cell factor 4 (Tcf7l2), the intestinal mediator of Wnt/ß-catenin-dependent transcription. The Wnt/ß-catenin dependence of Stat3 activity in the gut is confirmed by abrupt expansion of Stat3-positive cells in intestinal adenomas of apc heterozygotes. Our findings indicate that Jak/Stat3 signalling is needed for intestinal stem cell maintenance and possibly crucial in controlling Wnt/ß-catenin-dependent colorectal cancer cell proliferation.
Assuntos
Mucosa Intestinal/metabolismo , Fator de Transcrição STAT3/metabolismo , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Intestinos/crescimento & desenvolvimento , Intestinos/fisiologia , Janus Quinase 1 , Larva/crescimento & desenvolvimento , Larva/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição SOX9/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , beta Catenina/metabolismoRESUMO
Common genetic variations in the gene encoding transcription factor 7-like 2 (TCF7L2) reveal the strongest association with type 2-diabetes known to date. These lead to impaired insulin production and output, but the mechanisms of disease remain incompletely known. In this issue of Diabetologia, two publications provide new insights into TCF7L2-dependent diabetes.
Assuntos
Glucose/metabolismo , Homeostase/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiologia , Pâncreas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Animais , Humanos , Secreção de InsulinaRESUMO
AIMS/HYPOTHESIS: Individuals carrying type 2 diabetes risk alleles in TCF7L2 display decreased beta cell levels of T cell factor 7 like-2 (TCF7L2) immunoreactivity, and impaired insulin secretion and beta cell sensitivity to glucagon-like peptide 1 (GLP-1). Here, we sought to determine whether selective deletion of Tcf7l2 in mouse pancreas impairs insulin release and glucose homeostasis. METHODS: Pancreas-specific Tcf7l2-null (pTcf7l2) mice were generated by crossing mice carrying conditional knockout alleles of Tcf7l2 (Tcf7l2-flox) with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1.Cre). Gene expression was assessed by real-time quantitative PCR and beta cell mass by optical projection tomography. Glucose tolerance, insulin secretion from isolated islets, and plasma insulin, glucagon and GLP-1 content were assessed by standard protocols. RESULTS: From 12 weeks of age, pTcf7l2 mice displayed decreased oral glucose tolerance vs control littermates; from 20 weeks they had glucose intolerance upon administration of glucose by the intraperitoneal route. pTcf7l2 islets displayed impaired insulin secretion in response to 17 (vs 3.0) mmol/l glucose (54.6 ± 4.6%, p < 0.01) or to 17 mmol/l glucose plus 100 nmol/l GLP-1 (44.3 ± 4.9%, p < 0.01) compared with control islets. Glp1r (42 ± 0.08%, p < 0.01) and Ins2 (15.4 ± 4.6%, p < 0.01) expression was significantly lower in pTcf7l2 islets than in controls. Maintained on a high-fat (but not on a normal) diet, pTcf7l2 mice displayed decreased expansion of pancreatic beta cell volume vs control littermates. No differences were observed in plasma insulin, proinsulin, glucagon or GLP-1 concentrations. CONCLUSIONS/INTERPRETATION: Selective deletion of Tcf7l2 in the pancreas replicates key aspects of the altered glucose homeostasis in human carriers of TCF7L2 risk alleles, indicating the direct role of this factor in controlling beta cell function.
