The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression.
The transcription factor Rfx3 regulates beta-cell differentiation, function, and glucokinase expression.
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作者：
Ait-Lounis A;Bonal C;Seguín-Estévez Q;Schmid CD;Bucher P;Herrera PL;Durand B;Meda P;Reith W
缺乏转录因子Rfx 3的围产期小鼠的胰岛表现出产生胰岛素的β细胞的显著减少。这项工作的目的是解开这种缺陷背后的细胞和分子机制。免疫荧光研究和定量RT-PCR实验用于研究胰岛素阳性细胞的出现，与内分泌祖细胞分化β细胞有关的转录因子的表达，以及Rfx 3 −/−和胰腺特异性Rfx 3基因敲除小鼠发育过程中成熟β细胞标志物的表达。进行RNA干扰实验以记录在Min 6 β细胞中下调Rfx 3表达的结果。定量染色质免疫沉淀（ChIP），ChIP测序和带移实验被用来确定Rfx 3靶基因。Rfx 3 −/−小鼠中胰岛素阳性细胞的发育减少不是由于内分泌祖细胞或β谱系特化缺陷，而是反映了胰岛素阳性β细胞前体和缺陷β细胞的积累，表现出胰岛素、Glut-2和Gck表达减少。类似的不完全分化的β细胞在胰腺特异性Rfx 3缺陷的胚胎中发育。缺乏Glut-2和Gck表达的缺陷β细胞在Rfx 3缺陷的成人中占主导地位，导致葡萄糖耐受不良。减弱的Glut-2和葡萄糖激酶的表达，受损的葡萄糖刺激的胰岛素分泌，也诱导RNA干扰介导的抑制Rfx 3在Min 6细胞中的表达。最后，发现Rfx 3在Min 6细胞和人类胰岛中与葡萄糖激酶基因的神经内分泌启动子中的两个众所周知的调节序列，即Rfx 1和Rfx 2结合。我们的研究结果表明，Rfx 3是成熟β细胞的分化和功能所必需的，并调节葡萄糖激酶基因的β细胞启动子。
Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing β-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency. Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of β-cells from endocrine progenitors, and the expression of mature β-cell markers during development in Rfx3−/− and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 β-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes. Reduced development of insulin-positive cells in Rfx3−/− mice was not due to deficiencies in endocrine progenitors or β-lineage specification, but reflected the accumulation of insulin-positive β-cell precursors and defective β-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated β-cells developed in pancreas-specific Rfx3-deficient embryos. Defective β-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference–mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene. Our results show that Rfx3 is required for the differentiation and function of mature β-cells and regulates the β-cell promoter of the glucokinase gene.
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