Abstract
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.
Highlights
Catenin alpha-like-1(CTNNAL1) was first characterized as a 2.45-kb transcript that was down-regulated in human pancreatic cancer cells [1]
The results showed that expression of catenin alpha-like 1 (CTNNAL1) protein was increased after ozone stress
By site-directed mutagenesis of putative transcriptionfactor-binding sites within pGL3/FR/luc, we observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2a and Lymphoid Enhancer binding Factor-1 (LEF-1) sites
Summary
Catenin alpha-like-1(CTNNAL1) was first characterized as a 2.45-kb transcript that was down-regulated in human pancreatic cancer cells [1]. With 734 amino acids, the predicted CTNNAL1 polypeptide has similarities to human vinculin and a-catenin, especially in the N-terminal region, which contains binding sites for b-catenin, talin and a-actinin [2]. Amphipathic helices in the C-terminal homology region corresponding to a-catenin contain potential binding sites for the tight junction protein ZO-1 and the actin cytoskeleton [2,3], suggesting that CTNNAL1 may act as a cytoskeletal linker protein. Wiesner C et al [7] reported that CTNNAL1 interacts with the IkB kinase (IKK)- b, a key component of the NF-kB signaling pathway. Ectopic expression of CTNNAL1 augmented NF-kB activity, promoted cell migration and increased cell resistance to apoptosis
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