Tan Yu-rong, Qin Xiao-qun, Xiang Yang, Yang Tao, Qu Fei, Wang Yue, Liu Hui-jun, Weber H Christian
Department of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan, China.
Biochem J. 2007 Jul 1;405(1):131-7. doi: 10.1042/BJ20061754.
Previously, we found that bombesin receptor subtype 3 (BRS-3) significantly increased in an ozone-stressed airway hyperresponsiveness animal model and resulted in induced wound repair and protection from acute lung injury. In the present study, we determined molecular mechanisms of BRS-3 regulation in human BECs (bronchial epithelial cells) in response to ozone stress. Ten oligonucleotide probes corresponding to various regions of the BRS-3 promoter were used in EMSA (electrophoretic mobilityshift assays). Four were found to have an enhanced mobility shift with extracts from ozone-stressed cells. On the basis of the assay of mutated probes binding with extracts and antibody supershift, they were verified as MTF-1 (metal-regulatory-element-binding transcription factor-1), PPARalpha (peroxisome-proliferator-activated receptor alpha), AP-2alpha (activator protein 2alpha) and HSF-1 (heat-shock factor 1). Next, ChIP (chromatin immunoprecipitation) assay, site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe these transcription factors associated with the BRS-3 promoter. Only AP-2alpha and PPARalpha increased ozone-inducible DNA binding on the BRS-3 promoter and BRS-3 expression. The time courses of AP-2alpha and PPARalpha activation, followed by BRS-3 expression, were also examined. It was shown that ozone-inducible BRS-3 expression and AP-2alpha- and PPARalpha-binding activity correlated over a 48 h period. The translocation of PPARalpha was observed by immunofluorescence assay, which showed that PPARalpha nuclear translocation increased after ozone exposure. Our data suggest that AP-2alpha and PPARalpha may be especially involved in this ozone-inducible up-regulation mechanism of BRS-3 expression.
此前，我们发现蛙皮素受体亚型3（BRS-3）在臭氧应激性气道高反应性动物模型中显著增加，并导致诱导伤口修复以及对急性肺损伤起到保护作用。在本研究中，我们确定了人支气管上皮细胞（BECs）中BRS-3在响应臭氧应激时的调控分子机制。在电泳迁移率变动分析（EMSA）中使用了与BRS-3启动子不同区域相对应的10种寡核苷酸探针。发现其中4种与臭氧应激细胞提取物的迁移率变动增强。基于对与提取物结合的突变探针和抗体超迁移的分析，它们被证实为金属调节元件结合转录因子-1（MTF-1）、过氧化物酶体增殖物激活受体α（PPARα）、激活蛋白2α（AP-2α）和热休克因子1（HSF-1）。接下来，使用染色质免疫沉淀（ChIP）分析、定点诱变技术和反义寡核苷酸技术来观察这些与BRS-3启动子相关联的转录因子。只有AP-2α和PPARα增加了BRS-3启动子上臭氧诱导的DNA结合以及BRS-3表达。还检测了AP-2α和PPARα激活以及随后BRS-3表达的时间进程。结果表明，在48小时内，臭氧诱导的BRS-3表达与AP-2α和PPARα结合活性相关。通过免疫荧光分析观察到PPARα的易位，结果表明臭氧暴露后PPARα核易位增加。我们的数据表明，AP-2α和PPARα可能特别参与了这种BRS-3表达的臭氧诱导上调机制。
