The Regulation Of CTNNAL1 Expression And Its Role On Bronchial Epithelial Cells And Asthma

Objectives:Destruction in airway epithelium structure and integrity,which is often induced by flaws of constitutive adhesion,is the essential link of asthma pathogenicity.During the course of AHR or airway inflammation, airway epithelial cells express adhesion molecules to arrest leucocytes and evoke an inflammation reaction in airway.Airway epithelial cells anchored to extracellular matrix through intergrins and sustained the ability of against injury,repair and integrity in structure or in function of human bronchial epithelial cells(BEC).Therefore it can be reasonably speculated that abnormal expression of adhesive molecules in airway epithelial cells, which might be possibly due to either correspond coding gene alteration or imbalance expression among various kind of adhesion molecules, contribute to aberrant susceptibility to asthma.Based on the result of asthma-associated adhesion molecules expression spectrum,CTNNAL1, whose expression was down regulated in asthma patients,was selected as a candidate in this present study for a further functional investigation.Up to now,there is very little known about the pulmonary biological effects, gene expression modulation of CTNNAL1.The present study was designed to identity the association of CTNNAL 1 with asthma,observe the pulmonary biological effects and gene expression modulation of CTNNAL1.Contents:1.Identity the association of CTNNAL 1 with asthmaOur previous study used a cDNA microarray to screen the differential expression of adhesion molecules in human peripheral blood leucocytes, and found that an alpha-catenin-related protein,catenin alpha-like 1 (CTNNAL1) was downregulated in asthma patients.In this study,Real time PCR was used to further confirm the expression of CTNNAL1 in asthm.Immunohistochemistry was used to verified CTNNAL1 expression on tunica mucosa bronchiorum of normal person and asthma patients. Using a well-characterized OVA-sensitized Balb/c mice asthma model,we carried out a time course study of the expression of CTNNAL1 in lung following the AHR progression.Results:In situ hybridizition showed CTNNAL1 was predominantly localized in the ciliated columnar epithelium of bronchioles,secretory cells of terminal bronchioles,vascular endothelial cells and alveolar cells.The expression of CTNNAL1 mRNA was significantly down regulated in the peripheral blood leucocyte and tunica mucosa bronchiorum.Real-time PCR showed the mRNA level of CTNNAL1 was decreased in asthma animals.There was a negative correlation between the pulmonary resistance(R_L) in asthma mice and the levels of CTNNAL1 mRNA.This present study demonstrated that CTNNAL1 is highly correlated with asthma.2.Function study of CTNNAL1 on airway epitheliumBalb/c mice were inhaled a mixture of 2.0ppm ozone and fresh air for one,two,four,and eight days respectively to establish a airway stressed animal model and the time and spatial expression of CTNNAL1 were examined by ISH and Real-time PCR.The results showed inflammationary infiltration,mucosal exudation,cavitary stricture and bronchial epithelial denudation were observed in the ozone-stressed group.The expression of CTNNAL1 mRNA was increased with the ozone stress,peaked on the second day,and then reduced.The results also showed that expression of CTNNAL1 mRNA was explicitly increased in ozone-stressed BEC.To investigate function of CTNNAL1 in asthma,we established a CTNNAL1 expression plasmid and designed CTNNAL1 antisense oligonucleotide(ASO) to promoter or hinder the expression of CTNNAL1. Then we use them to observe the effect of CTNNAL1 on the repair and proliferation of BEC.A small wound was made in the confluent monolayer by mechanical scraping and the remaining wound area was measured serially per 4 hours in 24h by video microscopy.A linear regression equation of the remaining wound area to time was obtained and the slope was used to judge the repair speed of BECs.The proliferation of BECs was assayed by MTT.The results showed that the wound repair and proliferation of BECs were accelerated by CTNNAL1. We also detect the effect of CTNNAL1,a member of vinculin family, on Fn mediated wound repair,cell proliferation and focal adhesion kinase phosphorylation.Western blot showed that Fn could promote FAK phosphorylation in a transient time-dependent manner.CTNNAL1 ASO alone did not cause FAK phosphorylation.However,it could inhibit FAK phosphorylation induced by Fn,indicating that CTNNAL1 might have a role in modulating migration and proliferation via FAK phosphorylation signal transduction from Fn.Our results showed a novel link between CTNNAL1 with Fn mediated cell-extracellular matrix adhesion.These results raise the possibility that the down regulation of CTNNAL1 might contribute to the asthma development because of the attenuated cell-cell and cell-matrix adhesion,which lead to the bronchial epithelium desquamated and asthma.3.Study gene expression modulation of CTNNAL1In the present study,we determined molecular mechanisms of CTNNAL1 regulation in human BEC.8 oligonucleotide probes corresponding to various regions of the CTNNAL1 promoter were used in EMSA(electrophoretic mobilityshift assays).5 were found to have an enhanced mobility shift with extracts from BECs.On the basis of the assay of mutated probes and antibody supershift,they were verified as LEF-1, AP-2αand CREB.Next,ChIP(chromatin immunoprecipitation) assay was used to observe the interaction between these transcription factors and CTNNAL1 promoter.Only AP-2αand LEF-1 show the binding on CTNNAL1 promoter.By site-directed mutagenesis of putative transcription-factor-binding sites within pGL3/CTNNAL1/luc,we observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2αand LEF-1 sites.Our data suggest that AP-2αand LEF-1 may be involved in regulation of CTNNAL1 expression.The time courses of AP-2αand LEF-1 activation,followed by CTNNAL1 expression were also examined.It was shown that ozone stress can activate the AP-2αand LEF-1 with one hour,ozone-inducible CTNNAL1 expression and AP-2αand LEF-1 binding activity correlated during a-16 hour time course. Conclusion:The present study demonstrates a robust transcriptional CTNNAL1 up-regulation occurs during acute ozone-induced stress and is mediated at least in part by ozone-induced recruitment of LEF-1 and AP-2αto the human CTNNAL1 promoter thereby accelerated wound repair and proliferation of BEC,which ameliorating the effects of lung injury.The down-regulation of CTNNAL1 in asthma patients or asthma animal model may lead to the bronchial epithelium desquamated and AHR.