Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells
Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells
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DOI：
10.1016/j.bbrc.2013.03.084
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发表时间：
2013-05-03
影响因子：
3.1
通讯作者：
Tanizawa, Yukio
中科院分区：
文献类型：
--
作者：
Nakabayashi, Hiroko;Ohta, Yasuharu;Tanizawa, Yukio
芳香烃受体核转位蛋白（ARNT）/缺氧诱导因子-1 β（HIF-1 β）已成为人类胰腺β细胞功能障碍和2型糖尿病的潜在决定因素。与非糖尿病供体相比，在来自2型糖尿病供体的胰岛中观察到Arnt表达减少82%。然而，Arnt表达的调控因子很少被鉴定。与此同时，已知生物钟成分CLOCK和BMAL 1的破坏会导致低胰岛素血症和糖尿病，但分子细节仍不清楚。在这项研究中，我们确定了Arnt和两个时钟控制的输出基因，白蛋白D-元件结合蛋白（Dbp）和E4结合蛋白4（E4 bp 4）之间的一种新的分子连接。通过使用Wfs 1（-/-）A（y）/a小鼠的胰岛进行基因表达研究，我们证明了糖尿病小鼠中时钟相关基因表达的改变。在Zeitgever时间（ZT）12时，DBP mRNA降低50%，E4 bp 4 mRNA增加50%，Arnt mRNA降低30%。小鼠胰岛表现出时钟基因表达的振荡。E4 BP 4是一个D盒负调节器，与DBP（一个D盒正调节器）反相振荡。我们还发现Arnt mRNA的低幅度昼夜节律表达，在ZT 4达到峰值。在HEK 293和MIN 6细胞系中，DBP的过表达使ARNT的mRNA和蛋白水平均升高。在MIN 6细胞中的Arnt启动子驱动的荧光素酶报告基因测定显示，DBP使Arnt启动子活性增加2.5倍，并且E4 BP 4竞争性抑制其激活。此外，在ChIP测定中，DBP和E4 BP 4直接结合MIN 6细胞中Arnt启动子内的D-box元件。这些结果表明，在小鼠胰岛中Arnt的mRNA表达以昼夜节律的方式显著波动，并且Dbp的下调和E4 bp 4的上调有助于糖尿病中Arnt表达的直接抑制。(C)2013 Elsevier Inc. All rights reserved.
Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1 beta (HIF-1 beta) has emerged as a potential determinant of pancreatic beta-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4).By conducting gene expression studies using the islets of Wfs1(-/-) A(y)/a mice that develop severe diabetes due to beta-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes. (C) 2013 Elsevier Inc. All rights reserved.