RESUMO
TGFß superfamily ligands, receptors, and second messengers, including activins A and B, have been identified in pancreatic islets and proposed to have important roles regulating development, proliferation, and function. We previously demonstrated that Fstl3 (an antagonist of activin activity) null mice have larger islets with ß-cell hyperplasia and improved glucose tolerance and insulin sensitivity in the absence of altered ß-cell proliferation. This suggested the hypothesis that increased activin signaling influences ß-cell expansion by destabilizing the α-cell phenotype and promoting transdifferentiation to ß-cells. We tested the first part of this hypothesis by treating α- and ß-cell lines and sorted mouse islet cells with activin and related ligands. Treatment of the αTC1-6 α cell line with activins A or B suppressed critical α-cell gene expression, including Arx, glucagon, and MafB while also enhancing ß-cell gene expression. In INS-1E ß-cells, activin A treatment induced a significant increase in Pax4 (a fate determining ß-cell gene) and insulin expression. In sorted primary islet cells, α-cell gene expression was again suppressed by activin treatment in α-cells, whereas Pax4 was enhanced in ß-cells. Activin treatment in both cell lines and primary cells resulted in phosphorylated mothers against decapentaplegic-2 phosphorylation. Finally, treatment of αTC1-6 cells with activins A or B significantly inhibited proliferation. These results support the hypothesis that activin signaling destabilized the α-cell phenotype while promoting a ß-cell fate. Moreover, these results support a model in which the ß-cell expansion observed in Fstl3 null mice may be due, at least in part, to enhanced α- to ß-cell transdifferentiation.
Assuntos
Ativinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Diferenciação Celular/genética , Linhagem Celular , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Células Cultivadas , Glucagon/efeitos dos fármacos , Glucagon/genética , Células Secretoras de Glucagon/metabolismo , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Insulina/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Fator de Transcrição MafB/efeitos dos fármacos , Fator de Transcrição MafB/genética , Masculino , Camundongos , Fatores de Transcrição Box Pareados/efeitos dos fármacos , Fatores de Transcrição Box Pareados/genética , Transativadores/efeitos dos fármacos , Transativadores/genética , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Adenosine monophosphate-activated protein kinase (AMPK) is stimulated in embryos during diabetic pregnancy by maternal hyperglycaemia-induced embryo oxidative stress. Stimulation of AMPK disrupts embryo gene expression and causes neural tube defects. Metformin, which may be taken during early pregnancy, has been reported to stimulate AMPK activity. Thus, the benefits of improved glycaemic control could be offset by stimulated embryo AMPK activity. Here, we investigated whether metformin can stimulate AMPK activity in mouse embryos and can adversely affect embryo gene expression and neural tube defects. METHODS: Pregnant nondiabetic mice were administered metformin beginning on the first day of pregnancy. Activation of maternal and embryo AMPK [phospho-AMPK α (Thr172) relative to total AMPK], expression of Pax3, a gene required for neural tube closure, and neural tube defects were studied. Mouse embryonic stem cells were used as a cell culture model of embryonic neuroepithelium to study metformin effects on AMPK and Pax3 expression. RESULTS: Metformin had no effect on AMPK in embryos or maternal skeletal muscle but increased activated AMPK in maternal liver. Metformin did not inhibit Pax3 expression or increase neural tube defects. However, metformin increased activated AMPK and inhibited Pax3 expression by mouse embryonic stem cells. Mate1/Slc47a1 and Oct3/Slc22a, which encode metformin transporters, were expressed at barely detectable levels by embryos. CONCLUSIONS: Although metformin can have effects associated with diabetic embryopathy in vitro, the lack of effects on mouse embryos in vivo may be due to lack of metformin transporters and indicates that the benefits of metformin on glycaemic control are not counteracted by stimulation of embryo AMPK activity and consequent embryopathy.
Assuntos
Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Gestacional/tratamento farmacológico , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/enzimologia , Metformina/efeitos adversos , Gravidez em Diabéticas/tratamento farmacológico , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Defeitos do Tubo Neural/induzido quimicamente , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/efeitos dos fármacos , GravidezRESUMO
CONCLUSION: Our results indicate that myosin VIIa-positive cells are generated from embryonic stem cells (ESCs) co-cultured with PA6 cells; however, bone morphogenetic protein 4 (BMP4) may not be a key molecule for induction of myosin VIIa-positive cells from the ESCs. BACKGROUND: ESCs have been considered as a basis for cell therapy in a range of organs, because of their potential for self-renewal and pluripotency. Co-culture with PA6 stromal cells can induce differentiation of ESCs into various types of ectodermal cells including sensory progenitors. BMP4 plays an essential role in the development of sensory hair cells in the inner ear. MATERIALS AND METHODS: We examined effects of BMP4 on differentiation of ESCs into the hair cell immunophenotype. BMP4 was supplemented at different time points to ESCs co-cultured on PA6 stromal cells. The ESCs were then collected and examined for the expression of myosin VIIa, a hair cell marker, and betaIII-tubulin, a neural marker. The expression of myosin VIIa and betaIII-tubulin was identified. RESULTS: Quantitative assessments revealed that exogenous BMP4 has significant effects on the expression of betaIII-tubulin, but not of myosin VIIa.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Dineínas/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Miosinas/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/administração & dosagem , Proteínas de Homeodomínio/efeitos dos fármacos , Camundongos , Miosina VIIa , Fatores de Transcrição Box Pareados/efeitos dos fármacos , Células-Tronco/efeitos dos fármacosRESUMO
The PAX4 gene, a member of the paired box (PAX) gene family, is thought to be involved in regulating the fate of beta-cells in the mammalian pancreas. We observed the aberrant expression of PAX4 mRNA in 10 of 15 hematologic cell lines analyzed by RT-PCR. The restoration of PAX4 gene expression after treatment with the demethylating agent 5-aza-2'-deoxycitidine, as well as bisulfite sequencing analysis, indicated that gene overexpression was caused by DNA demethylation at the promoter region. Such DNA demethylation also was observed in primary lymphoma (20 out of 45 patients) on combined bisulfite restriction assay (COBRA). Forced expression of the PAX4 gene in the HEK293 and SHSY/610 cell lines conferred positive effects on cell growth. This profile of PAX4 thus corresponds to that of a candidate oncogene in hematologic malignancies.