Look D C, Pelletier M R, Holtzman M J
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1994 Mar 25;269(12):8952-8.
Intercellular adhesion molecule-1 (ICAM-1) modulates epithelial and endothelial leukocyte adherence, but epithelial cell ICAM-1 levels (unlike endothelial cell levels) are selectively sensitive to interferon-gamma (IFN-gamma). Nuclear run-off assays indicated that IFN-gamma regulation of epithelial ICAM-1 levels occurs at a transcriptional level, so the basis for selective cytokine control of ICAM-1 expression was investigated using the ICAM-1 gene promoter region. A plasmid construct containing 5.0 kilobases of ICAM-1 gene 5'-flanking region fused to a reporter gene was selectively responsive to IFN-gamma in (human tracheal) epithelial cells but not in (human umbilical vein) endothelial cells, indicating that this region is sufficient to mediate proper cell-specific expression. An IFN-gamma response element (IRE) was localized to a DNA segment (nucleotides -130 to -94) in the ICAM-1 gene by comparisons of nested 5'-deletional constructs and by demonstrating that this segment confers IFN-gamma responsiveness on heterologous promoters. This same IRE formed a single major binding complex (IRE-BC) in gel retardation assays with nuclear proteins from IFN-gamma-stimulated but not unstimulated epithelial cells, and mutation of the IRE consensus motif (TTTCCGGGAAA at -116 to -106) resulted in loss of IRE-protein binding and abolished IFN-gamma responsiveness, indicating that this sequence is required for ICAM-1 IRE function. Comparison of the ICAM-1 IRE to DNA elements that confer IFN responsiveness in other human genes indicated similarity only to response regions in the Fc gamma RI and IRF-1 genes. The findings provide evidence for a distinct IRE subset that combines a DNA element common to all IFN-responsive genes (GAAA) with a distinct flanking sequence (the inverted repeat GAAA) in order to fine-tune IFN responses and activate a subset of immune response genes (ICAM-1, Fc gamma RI, and IRF-1).
细胞间黏附分子-1（ICAM-1）调节上皮细胞和内皮细胞与白细胞的黏附，但上皮细胞ICAM-1水平（与内皮细胞水平不同）对干扰素-γ（IFN-γ）具有选择性敏感性。核转录分析表明，IFN-γ对上皮细胞ICAM-1水平的调节发生在转录水平，因此使用ICAM-1基因启动子区域研究了ICAM-1表达的选择性细胞因子控制基础。一种含有5.0千碱基ICAM-1基因5'-侧翼区域并与报告基因融合的质粒构建体，在（人气管）上皮细胞中对IFN-γ有选择性反应，但在（人脐静脉）内皮细胞中无反应，表明该区域足以介导适当的细胞特异性表达。通过比较嵌套的5'-缺失构建体，并证明该片段赋予异源启动子IFN-γ反应性，将IFN-γ反应元件（IRE）定位到ICAM-1基因中的一个DNA片段（核苷酸-130至-94）。在凝胶阻滞试验中，这个相同的IRE与来自IFN-γ刺激而非未刺激的上皮细胞核蛋白形成单一主要结合复合物（IRE-BC），并且IRE共有基序（-116至-106处为TTTCCGGGAAA）的突变导致IRE-蛋白结合丧失并消除了IFN-γ反应性，表明该序列是ICAM-1 IRE功能所必需的。将ICAM-1 IRE与赋予其他人类基因IFN反应性的DNA元件进行比较，发现仅与FcγRI和IRF-1基因中的反应区域相似。这些发现为一个独特的IRE亚群提供了证据，该亚群将所有IFN反应性基因共有的DNA元件（GAAA）与一个独特的侧翼序列（反向重复GAAA）结合起来，以微调IFN反应并激活一部分免疫反应基因（ICAM-1、FcγRI和IRF-1）。
