Pettersson A T, Laurencikiene J, Nordström E A, Stenson B M, van Harmelen V, Murphy C, Dahlman I, Rydén M
Department of Medicine, Huddinge, Lipid Laboratory, Novum, Karolinska Institutet, Stockholm, Sweden.
Int J Obes (Lond). 2008 Sep;32(9):1380-7. doi: 10.1038/ijo.2008.101. Epub 2008 Jul 8.
Cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) is a protein that regulates lipolysis in human adipocytes through cross-talk involving tumor necrosis factor-alpha (TNF-alpha). TNF-alpha downregulates CIDEA mRNA although it is unclear whether this is mediated through transcriptional or post-transcriptional mechanisms. CIDEA has important metabolic effects in human fat cells and genetic variations in the human CIDEA gene have been correlated to the development of obesity. However, little is known about the factors regulating CIDEA expression in human adipocytes. We set out to describe the transcriptional control of human CIDEA.
A 1.1-kb genomic fragment upstream of the transcriptional start site (TSS) of human CIDEA was cloned and deletion fragments were generated. Transcriptional activity of the promoter was analyzed by luciferase reporter assays in in vitro-differentiated human adipocytes. The effect of TNF-alpha was assessed in human adipocytes and murine 3T3-L1 cells transfected with deletion fragments of the CIDEA promoter. Protein-DNA interactions were analyzed by electrophoretic mobility shift assays (EMSA).
Basal transcriptional activity was found in a 97-bp region upstream of the TSS. We studied the effect of three common haplotypes in the promoter region but found no significant difference in transcriptional activity among them. Incubation of in vitro-differentiated human adipocytes as well as 3T3-L1 cells with TNF-alpha reduced the transcriptional activity of the human CIDEA promoter, demonstrating a direct effect on CIDEA transcription. EMSAs and mutational analysis indicated that this was mediated by a nuclear factor-kappaB (NF-kappaB) site at position -163/-151.
We demonstrate that basal transcription of the human CIDEA gene is confined to the 97 first bases upstream of TSS and that TNF-alpha negatively regulates transcription of this gene, which at least in part involves NF-kappaB activation.
细胞死亡诱导DFFA（DNA片段化因子α）样效应因子A（CIDEA）是一种通过涉及肿瘤坏死因子α（TNF-α）的相互作用来调节人类脂肪细胞脂解作用的蛋白质。TNF-α会下调CIDEA mRNA，但其是否通过转录或转录后机制介导尚不清楚。CIDEA在人类脂肪细胞中具有重要的代谢作用，人类CIDEA基因的遗传变异与肥胖症的发生有关。然而，关于调节人类脂肪细胞中CIDEA表达的因素知之甚少。我们着手描述人类CIDEA的转录调控。
克隆了人类CIDEA转录起始位点（TSS）上游1.1 kb的基因组片段，并生成了缺失片段。通过荧光素酶报告基因检测在体外分化的人类脂肪细胞中分析启动子的转录活性。在转染了CIDEA启动子缺失片段的人类脂肪细胞和小鼠3T3-L1细胞中评估TNF-α的作用。通过电泳迁移率变动分析（EMSA）分析蛋白质-DNA相互作用。
在TSS上游97 bp区域发现了基础转录活性。我们研究了启动子区域三种常见单倍型的作用，但发现它们之间的转录活性没有显著差异。用TNF-α孵育体外分化的人类脂肪细胞以及3T3-L1细胞会降低人类CIDEA启动子的转录活性，表明对CIDEA转录有直接影响。EMSA和突变分析表明，这是由位于-163/-151位置的核因子κB（NF-κB）位点介导的。
我们证明人类CIDEA基因的基础转录局限于TSS上游的前97个碱基，并且TNF-α负调节该基因的转录，这至少部分涉及NF-κB激活。
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