Identification of transcription factors regulating CTNNAL1 expression in human bronchial epithelial cells.
Identification of transcription factors regulating CTNNAL1 expression in human bronchial epithelial cells.
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人支气管上皮细胞中调节 CTNNAL1 表达的转录因子的鉴定
DOI：
10.1371/journal.pone.0031158
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Liu CX
中科院分区：
文献类型：
--
作者：
Xiang Y;Qin XQ;Liu HJ;Tan YR;Liu C;Liu CX
粘附分子在气道高反应性或气道炎症中起重要作用。本课题组前期研究表明，α-连环蛋白相关蛋白--连环蛋白α样1（CTNNAL 1）在哮喘患者和动物模型中表达下调。在这项研究中，我们观察到CTNNAL 1的表达增加，在肺组织的臭氧应激Balb/c小鼠模型和急性臭氧应激的人支气管上皮细胞（HBEC）。为了鉴定CTNNAL 1基因在HBEC中转录的可能的DNA结合蛋白，我们设计了8个寡核苷酸探针，分别对应于CTNNAL 1启动子的不同区域，采用电泳迁移率变动分析（EMSA）。通过EMSA和抗体超移位实验，我们在CTNNAL 1启动子区域发现了5个可能的转录因子结合位点，分别可以募集LEF-1、AP-2α和CREB。染色质免疫沉淀（ChIP）实验证实AP-2 α和LEF-1可以被募集到CTNNAL 1启动子上。因此，我们通过定点突变pGL 3/FR/luc中的AP-2和LEF-1位点，进一步分析CTNNAL 1启动子中这些位点的功能。我们观察到AP-2α和LEF-1位点突变体的人CTNNAL 1启动子活性降低。以LEF-1和AP-2α为靶点的ASO预处理显著降低了臭氧胁迫诱导的CTNNAL 1表达。AP-2α和LEF-1的激活以及CTNNAL 1的表达在16小时的时间过程中显示出相关性。我们的数据表明，一个强大的转录CTNNAL 1上调发生在急性臭氧诱导的压力，并介导至少部分由臭氧诱导的招募LEF-1和AP-2α的人CTNNAL 1启动子。
Adhesion molecules play important roles in airway hyperresponsiveness or airway inflammation. Our previous study indicated catenin alpha-like 1 (CTNNAL1), an alpha-catenin-related protein, was downregulated in asthma patients and animal model. In this study, we observed that the expression of CTNNAL1 was increased in lung tissue of the ozone-stressed Balb/c mice model and in acute ozone stressed human bronchial epithelial cells (HBEC). In order to identify the possible DNA-binding proteins regulating the transcription of CTNNAL1 gene in HBEC, we designed 8 oligo- nucleotide probes corresponding to various regions of the CTNNAL1 promoter in electrophoretic mobility shift assays (EMSA). We detected 5 putative transcription factors binding sites within CTNNAL1 promoter region that can recruit LEF-1, AP-2α and CREB respectively by EMSA and antibody supershift assay. Chromatin immunoprecipitation (ChIP) assay verified that AP-2 α and LEF-1 could be recruited to the CTNNAL1 promoter. Therefore we further analyzed the functions of putative AP-2 and LEF-1 sites within CTNNAL1 promoter by site-directed mutagenesis of those sites within pGL3/FR/luc. We observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2α and LEF-1 sites. Pre-treatment with ASOs targeting LEF-1and AP-2α yielded significant reduction of ozone-stress-induced CTNNAL1 expression. The activation of AP-2α and LEF-1, followed by CTNNAL1 expression, showed a correlation during a 16-hour time course. Our data suggest that a robust transcriptional CTNNAL1 up-regulation occurs during acute ozone-induced stress and is mediated at least in part by ozone-induced recruitments of LEF-1 and AP-2α to the human CTNNAL1 promoter.
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