Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products
Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products
复制标题
DOI：
10.1074/jbc.272.26.16498
复制
发表时间：
1997-06-27
影响因子：
4.8
通讯作者：
Schmidt, AM
中科院分区：
文献类型：
--
作者：
Li, JF;Schmidt, AM
晚期糖基化终末产物受体，β-葡聚糖，是细胞表面分子免疫球蛋白超家族的成员，在一系列细胞类型上差异表达。配体的连接扰乱了体内平衡机制，并可能为配体积累的病理情况下的细胞功能障碍提供了基础。为了了解潜在的表达的因素，我们克隆了5 '侧翼区的cDNA 3基因和其特征推定的调控基序。推定的启动子区域的分析显示存在三个潜在的NF-κ B样和两个SP1结合位点。使用与荧光素酶报告基因连接的嵌合5 '-缺失构建体瞬时转染血管内皮和平滑肌细胞，揭示了区域-1543/-587对基础和受刺激的cR基因表达都有重要贡献。这一区域的ESTA基因含有三个推定的NF-κ B样结合位点，并负责增加荧光素酶活性时观察到的内皮细胞或平滑肌细胞用脂多糖刺激。DNA酶I足迹分析和电泳迁移率变动分析显示，三个NF-κ B样结合位点中的两个（1和2）可能是功能性的，并对刺激有反应。在NF-κ B样位点1和2同时突变后，基础启动子表达和对LPS刺激的反应（通过相对荧光素酶活性测定）均显著减少。这些结果表明，NF-κ B依赖性机制调节细胞表达的炎症反应，并提出了一种手段，连接炎症反应。
The receptor for advanced glycation end products, RAGE, is a member of the immunoglobulin superfamily of cell surface molecules differentially expressed on a range of cell types. Ligation of RAGE perturbs homeostatic mechanisms and, potentially, provides a basis for cellular dysfunction in pathologic situations in which its ligands accumulate. To understand factors underlying RAGE expression, we cloned the 5'-flanking region of the RAGE gene and characterized putative regulatory motifs. Analysis of the putative promoter region revealed the presence of three potential NF-kappa B-like and two SP1 binding sites. Transient transfection of vascular endothelial and smooth muscle cells using chimeric 5'-deletion constructs linked to luciferase reporter revealed that the region -1543/-587 contributed importantly to both basal and stimulated expression of the RAGE gene. This region of the RAGE gene contained three putative NF-kappa B-like binding sites and was responsible for increased luciferase activity observed when endothelial or smooth muscle cells were stimulated with lipopolysaccharide. DNase I footprinting assays and electrophoretic mobility shift assay revealed that two of the three NF-kappa B-like binding sites (1 and 2) were likely functional and responsive to stimuli. Upon simultaneous mutation of NF-kappa B-like sites 1 and 2, both basal promoter expression and response to stimulation with LPS, as measured by relative luciferase activity, were significantly diminished. These results point to NF-kappa B-dependent mechanisms regulating cellular expression of RAGE and suggest a means of linking RAGE to the inflammatory response.