Kawai M, Nishikomori R, Jung E Y, Tai G, Yamanaka C, Mayumi M, Heike T
Department of Pediatrics, Faculty of Medicine, Kyoto University, Japan.
J Immunol. 1995 Mar 1;154(5):2333-41.
Intercellular adhesion molecule-1 (ICAM-1), the ligand of lymphocyte function-associated antigen-1, plays an important role in the interactions of a variety of hemopoietic and nonhemopoietic cells, including leukocytes, fibroblasts, and endothelial cells. ICAM-1 is known to be involved in the onset of several diseases such as inflammation, allograft rejection, and so on. In this report, we investigated the effects of dexamethasone, cyclosporin A, FK506, and pyrrolidine dithiocarbamate (PDTC) on the induction of the ICAM-1 gene by cytokines in fibroblasts. PDTC, a potent inhibitor of NF-kappa B, was shown by ELISA and FACS analysis to prevent dramatically the expression of the ICAM-1 gene stimulated by IL-1 alpha, IFN-gamma, and PMA, although the other reagents inhibited it only slightly. Ribonuclease protection assay revealed that PDTC blocked the expression of the ICAM-1 gene at the mRNA level. To elucidate the mechanism of this inhibition, we constructed a series of ICAM-1 promoter deletion mutants linked to the chloramphenicol acetyl transferase gene and analyzed the effect of PDTC on their activities. Transient transfection analysis indicated that the critical region for inhibition by PDTC is an NF-kappa B binding site-like motif (GGGAGGATTCC, ICAM-1 kappa B) that is located at position-540. Electrophoresis mobility shift assay revealed that PDTC actually inhibits the binding of NF-kappa B (or NF-kappa B-like) protein to the ICAM-1 kappa B site. These findings suggest that PDTC inhibits ICAM-1 gene expression by inhibiting the association of NF-kappa B (or NF-kappa B-like) protein with the ICAM-1 kappa B site.
细胞间黏附分子-1（ICAM-1）是淋巴细胞功能相关抗原-1的配体，在包括白细胞、成纤维细胞和内皮细胞在内的多种造血和非造血细胞的相互作用中发挥重要作用。已知ICAM-1参与多种疾病的发生，如炎症、同种异体移植排斥等。在本报告中，我们研究了地塞米松、环孢素A、FK506和吡咯烷二硫代氨基甲酸盐（PDTC）对成纤维细胞中细胞因子诱导ICAM-1基因的影响。ELISA和FACS分析显示，NF-κB的强效抑制剂PDTC可显著阻止IL-1α、IFN-γ和PMA刺激的ICAM-1基因表达，而其他试剂仅略有抑制作用。核糖核酸酶保护试验表明，PDTC在mRNA水平上阻断了ICAM-1基因的表达。为了阐明这种抑制机制，我们构建了一系列与氯霉素乙酰转移酶基因相连的ICAM-1启动子缺失突变体，并分析了PDTC对其活性的影响。瞬时转染分析表明，PDTC抑制的关键区域是位于-540位置的一个类似NF-κB结合位点的基序（GGGAGGATTCC，ICAM-1 κB）。电泳迁移率变动分析表明，PDTC实际上抑制了NF-κB（或类NF-κB）蛋白与ICAM-1 κB位点的结合。这些发现表明，PDTC通过抑制NF-κB（或类NF-κB）蛋白与ICAM-1 κB位点的结合来抑制ICAM-1基因表达。
