Involvement of CCAAT/enhancer-binding protein and nuclear factor-kappa B binding sites in interleukin-6 promoter inhibition by estrogens
Involvement of CCAAT/enhancer-binding protein and nuclear factor-kappa B binding sites in interleukin-6 promoter inhibition by estrogens
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DOI：
10.1210/me.10.6.713
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发表时间：
1996-06-01
影响因子：
--
通讯作者：
Garcia, T
中科院分区：
文献类型：
--
作者：
Galien, R;Evans, HF;Garcia, T
在绝经后妇女中观察到的骨丢失明显与雌激素水平的降低有关。白细胞介素6（IL-6）是一种多功能的细胞因子，参与破骨细胞的分化，由成骨细胞分泌，似乎是一个关键的分子，在破骨细胞的分化过程中。由于先前的报道表明人IL-6启动子被雌二醇抑制，我们研究了雌二醇（E2）介导的人细胞IL-6抑制的机制。使用IL-6 ELISA试验分析成骨细胞瘤Saos-2细胞中IL-6分泌作为时间的函数，结果表明，在处理2至24小时之间可以监测到E（2）对肿瘤坏死因子-α（TNF α）诱导的最大抑制。在Saos-2和MCF 7细胞中，IL-6抑制明显具有雌激素激动剂特异性。用pIL-6/CAT质粒和雌激素受体（人ER）表达载体瞬时转染HeLa细胞，证实了人ER在抑制IL-6启动子中的作用。启动子的缺失和突变分析强调了-185/-60区域的作用，并表明在MCF 7和HeLa细胞中，核因子-IL 6（NF-IL 6）位点与核因子-kappa B合作（NF-κ B）基序产生TNF α的最大诱导，而CCAAT/增强子结合蛋白（C/EBP）位点对NF-κ B显示出不同的协同作用，这取决于所用的细胞系。在HeLa细胞中，而不是在MCF 7细胞中，我们确定了C/EBP位点的重要作用，表明E（2）的敏感性明显依赖于其完整性。在这些细胞系中，NF-κ B位点突变消除了构建体的TNF α和E（2）敏感性。
Bone loss observed in postmenopausal women is clearly associated with a decrease in estrogen levels. Interleukin 6 (IL-6), a multifunctional cytokine involved in osteoclast differentiation, is secreted by osteoblasts and appears to be a key molecule in the osteoporotic process. As previous reports have shown that the human IL-6 promoter is inhibited by estradiol, we investigated the mechanism of estradiol (E(2))-mediated IL-6 inhibition in human cells. Analysis of the IL-6 secretion as a function of time in osteoblastoma Saos-2 cells, using an IL-6 ELISA test, showed that a maximal E(2) inhibition of tumor necrosis factor-alpha (TNF alpha) induction could be monitored between 2 and 24 h of treatment. IL-6 inhibition was clearly estrogen agonist-specific in Saos-2 and MCF7 cells. Transient transfections of HeLa cells with a pIL-6/CAT plasmid and an estrogen receptor (human ER) expression vector, confirmed the role of the human ER in inhibition of the IL-6 promoter. Deletion and mutational analysis of the promoter highlighted the role of the -185/-60 region and showed that in both MCF7 and HeLa cells, the nuclear factor-IL 6 (NF-IL6) site cooperates with the nuclear factor-kappa B (NF-kappa B) motif to produce maximal induction by TNF alpha, whereas the CCAAT/enhancer-binding protein (C/EBP) site displayed different cooperative effects toward NF-kappa B depending on the cell line used. In HeLa cells, but not in MCF7 cells, we defined an essential role for the C/EBP site by showing that the E(2) sensitivity was clearly dependent on its integrity. In these cell lines, the NF-kappa B site mutation abrogated both the TNF alpha- and E(2)- sensitivity of the construct.