Müller S, Kammerbauer C, Simons U, Shibagaki N, Li L J, Caughman S W, Degitz K
Department of Dermatology, Ludwig-Maximilians University, München, Germany.
J Invest Dermatol. 1995 Jun;104(6):970-5. doi: 10.1111/1523-1747.ep12606225.
The surface glycoprotein intercellular adhesion molecule-1 (ICAM-1) mediates important immunologic cell interactions during cutaneous inflammatory processes by binding to the leukocyte integrin lymphocyte function-associated antigen-1. The expression of ICAM-1 is induced in epidermal keratinocytes by certain pro-inflammatory stimuli, and this modulation is transcriptionally regulated. To identify the molecular mechanisms involved in the regulation of ICAM-1 gene expression, we have previously cloned the transcriptional regulatory region of the human ICAM-1-gene and have characterized a functional promoter. Here we have used the phorbol ester phorbol-12-myristate-13-acetate (PMA) to further evaluate the transcriptional mechanisms of ICAM-1 gene induction in A431 cells. Exposure to PMA induced ICAM-1 both at the mRNA and cell surface level. Promoter activity and PMA-enhanced effects were assessed by transiently transfecting A431 cells with chloramphenicol acetyl transferase reporter gene constructs containing a series of sequential ICAM-1 5' deletions. Constructs containing ICAM-1 5' fragments from -1162/+1 (relative to the transcription start site) to -277/+1 displayed a threefold increase in promoter activity when cells were stimulated with PMA. Inducibility dropped below 1.5-fold in chloramphenicol acetyl transferase construct -182/+1. Using electrophoretic mobility shift assays, a PMA-inducible binding site was identified for an NF kappa B-like complex within positions -186/-177. A -199/-170 fragment containing this NF kappa B-like element conferred PMA responsiveness when cloned into a thymidine kinase-driven chloramphenicol acetyl transferase vector, indicating that the region containing this NF kappa B-like element is not only necessary but also sufficient for PMA induction of ICAM-1.
表面糖蛋白细胞间黏附分子-1（ICAM-1）通过与白细胞整合素淋巴细胞功能相关抗原-1结合，在皮肤炎症过程中介导重要的免疫细胞相互作用。ICAM-1的表达由某些促炎刺激在表皮角质形成细胞中诱导，并且这种调节是转录调控的。为了确定参与ICAM-1基因表达调控的分子机制，我们之前克隆了人ICAM-1基因的转录调控区域并鉴定了一个功能性启动子。在此，我们使用佛波酯佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）进一步评估A431细胞中ICAM-1基因诱导的转录机制。暴露于PMA在mRNA和细胞表面水平均诱导了ICAM-1。通过用含有一系列连续ICAM-1 5'缺失的氯霉素乙酰转移酶报告基因构建体瞬时转染A431细胞来评估启动子活性和PMA增强的效应。当用PMA刺激细胞时，含有从-1162 / +1（相对于转录起始位点）到-277 / +1的ICAM-1 5'片段的构建体显示启动子活性增加了三倍。在氯霉素乙酰转移酶构建体-182 / +1中，诱导性降至1.5倍以下。使用电泳迁移率变动分析，在-186 / -177位置内鉴定了一个NFκB样复合物的PMA诱导性结合位点。当克隆到胸苷激酶驱动的氯霉素乙酰转移酶载体中时，含有该NFκB样元件的-199 / -170片段赋予了PMA反应性，表明含有该NFκB样元件的区域不仅对PMA诱导ICAM-1是必需的，而且也是充分的。
