CCSP A38G polymorphism in airway epithelium affects CCSP expression

Abstract

Background Club cell secretory protein (CCSP) is an endogenous anti-inflammatory product supposed to have an important protective role in the respiratory tract. Genome-wide association study and disease severity and susceptibility studies identified the A38G polymorphism in this gene as a candidate single-nucleotide polymorphism (SNP). Impaired airway homeostasis in chronic bronchial diseases (bronchiolitis obliterans syndrome, chronic obstructive pulmonary disease and asthma) might be related to CCSP defect but the SNP-transcription relationship is not established. This study was conducted to examine the influence of ccsp A38G polymorphism on CCSP expression at rest and to identify potentially modulating transcription factors. Methods Vectors constructions (proximal promoter: 229bp; and distal promoter: 926bp) containing -38G wild type or the -38A variant were designed. Human bronchial BEAS-2B cells were transfected with these proximal or distal promoters bearing wild type or mutant constructions. Dual luciferase assay system reflected CCSP transcription levels. In parallel, dual transfections with candidate modulating transcription factors identified by in silico analysis using ConSite and TFSEARCH were conducted. Results Transiently transfected BEAS-2B cells with the -38A 926bp variant vector induced less CCSP transcripts than the -38G and -38A 226bp vectors ( P -values 0.0256 and 0.0360, respectively). Cotransfection with Nkx2.1 (a candidate transcription factor overexpressed in most lung diseases with several binding sites identified by the in silico analysis within the CCSP gene promoter) significantly increased CCSP transcription. Interestingly, with proximal promoter only, -38A variant gene response to Nkx2.1 was higher than -38G wild type response. In depth-in silico analyzes identified two novel candidate transcription factors that could specifically bind to the A38G locus: Thing1E47 and p53. Further experiments will be done to check whether A38G polymorphism will differently affect CCSP production with these transcription factors. Conclusion Our data indicate that the distal promoter is the site bearing regulatory elements required for A38G polymorphism to affect CCSP expression. Also, -38A variant CCSP gene modulation induced by Nkx2.1 is significant. Finding transcription factors that could restore normal levels of CCSP expression is of interest.