Identification of Transcription Factors Regulating CTNNAL1 Expression in Human Bronchial Epithelial Cells

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

Growth factor and cytokine expression of human mesenchymal stromal cells is not altered in an in vitro model of tissue damage

Background aims. The beneficial effect of human (h) mesenchymal stromal cell (MSC) transplantation in a variety of cell-based intervention strategies is widely believed to be because of paracrine mechanisms. The modification of hMSC cytokine and growth-factor expression patterns were studied following exposure to lipopolysaccharide (LPS) and tissue homogenates (representing tissue debris) from normal and pathologic tissues. Methods. Human bone marrow-derived MSC were stimulated with LPS or exposed to homogenate from normal or pathologic rat spinal cord or heart. The expression profiles of a number of cytokines and growth factors were investigated using quantitative reverse transcription (RT)-polymerase chain reaction (PCR) with human-specific primers. The effects of tissue homogenates on hMSC proliferation and migratory behavior were also investigated. Results. Stimulation of hMSC with LPS resulted in an up-regulation of interleukin (IL)-1 beta, IL-6 and IL-8. However, the pattern of up-regulation varied between donor samples. Furthermore, LPS treatment resulted in a donor-dependent alteration of growth factor expression. Induction of a shift in expression pattern was not observed following exposure to homogenates from either normal or pathologic tissues. Tissue homogenates did stimulate cell proliferation, but not migration. Conclusions. The hMSC expression pattern is apparently stable, even when cells are confronted by debris from different tissue types. However, treatment of hMSC with LPS is able to change the expression of cytokines and growth factors in a donor-dependent manner that may enhance their potential use in regenerative medicine