The Oct DNA motif participates in the alcohol inhibition of the inducible nitric oxide synthase gene promoter in rat C6 glioma cells.
The Oct DNA motif participates in the alcohol inhibition of the inducible nitric oxide synthase gene promoter in rat C6 glioma cells.
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Oct DNA 基序参与大鼠 C6 神经胶质瘤细胞中诱导型一氧化氮合酶基因启动子的酒精抑制。
DOI：
10.1016/j.brainres.2007.08.047
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Syapin,PeterJ
中科院分区：
文献类型：
--
作者：
Sanchez,AlmaC;Davis,RandallL;Syapin,PeterJ
细胞因子和细菌产物诱导的一氧化氮合酶-2(INOS)与NOS2基因启动子近端和上游增强子区域的蛋白结合有关。为了阐明乙醇是如何抑制大鼠iNOS活性的，我们构建了NOS2启动子与荧光素酶基因融合的缺失突变体，并将其导入C6神经胶质细胞。稳定表达的细胞急性乙醇暴露24小时可抑制NOS2启动子的诱导活性，该启动子含有5‘侧翼区的缺失，包括一个94个碱基的启动子，该启动子缺乏任何已知的NF-κB位点，但携带一个C/EBpβ以及重叠的γ-IRE、GAS和OCT基序。乙醇未能抑制一个较小的78kb启动子的内源活性，该启动子缺乏C/eBPβ和重叠的γ-IRE和GAS基序，并且没有诱导活性。作为另一种方法，体内DNA足迹被用来鉴定诱导细胞中NOS2近端启动子的五个区域的蛋白质保护。急性乙醇暴露减少了5个启动子区域的蛋白质占有率，包括γ-IRE/NF-κB和重叠的γ-IRE/GAS/OCT位点。用1002bp的启动子对γ-IRE/GAS/OCT基序的八聚体结构域进行定点突变，研究其在乙醇抑制细胞因子和内毒素诱导活性中的作用。数据表明，当OCT基序缺失时，乙醇不能抑制启动子的活性。使用包含重叠的γ-IRE/GAS/OCT位点的22聚体探针进行的电泳迁移率改变分析显示，三个复合体与其中一个复合体被八聚体-1抗体竞争。这些观察结果表明，核心启动子上的蛋白质-DNA结合以及八聚体基序可能参与了乙醇对细胞因子和内毒素诱导的iNOS表达的调控。
Induction of nitric oxide synthase-2 (iNOS) by cytokines and bacterial products is associated with protein binding at the proximal promoter and in an upstream enhancer region of the Nos2 gene. To clarify how ethanol suppresses rat iNOS activity, we constructed several deletion mutants of the Nos2 promoter fused to the luciferase gene and transfected the constructs into C6 glial cells. Acute ethanol exposure of stably transfected cells for 24 h inhibits induced activity of Nos2 promoter constructs containing deletions in the 5′ flanking region, including a 94 bp promoter that lacks any known NF-κB site but which carries a C/EBPβ and overlapping γ-IRE, GAS and Oct motifs. Ethanol failed to inhibit the endogenous activity of a smaller, 78 bp promoter that lacks the C/EBPβ and overlapping, γ-IRE and GAS motifs and showed no inducible activity. As another approach, in vivo DNA footprinting was used and identified protein protections at five regions of the proximal Nos2 promoter in induced cells. Exposure to acute ethanol diminished protein occupation in the five promoter regions including the γ-IRE/NF-κB and the overlapping γ-IRE/GAS/Oct sites. Site-directed mutagenesis in the octamer domain of the γ-IRE/GAS/Oct motifs was studied in a 1002 bp promoter to examine its role in ethanol inhibition of cytokine and lipopolysaccharide induced activity. The data indicate that ethanol failed to inhibit promoter activity when the Oct motif is missing. Electrophoretic mobility shift assays performed using a 22-mer probe containing the overlapping γ-IRE/GAS/Oct sites showed three complexes with one of the complexes being competed by an octamer-1 antibody. These observations demonstrate the role of protein–DNA binding at the core promoter, and the likely involvement of the octamer motif, in ethanol modulation of cytokine and lipopolysaccharide induced iNOS expression.