Mechanisms of Glucocorticoid‐mediated Induction of Krüppel‐Like Factor 9 in the Human Airways
Authors: Mostafa Mahmoud M , Gerber Antony N , Newton Robert 
Published: June 2021

RATIONALEGlucocorticoids (GCs) are stress hormones that act on the GC receptor (GR; NR3C1) to elicit effects, including repression of inflammatory gene expression. However, the molecular mechanisms for this repressive effect remain unclear. Since GC, as inhaled corticosteroids, are the cornerstone of asthma treatment, a comparative transcriptome analysis of human airway epithelial cell variants was performed following GC treatment. This revealed 17 genes that were induced in common by GC. Of these, 7 genes were associated with gene ontology terms for transcriptional regulation. Many of these genes, including KLF9, were also induced by inhaled GC in vivo in the human airways. While, KLF9 is a probable repressor of transcription and has been implicated in cellular proliferation and differentiation, little is known about the GC‐mediated regulation of KLF9 in the airways. The current work addresses this knowledge gap.RESULTSGC‐mediated induction of KLF9 mRNA was confirmed in the airway epithelial cell lines, pulmonary type II A549 and bronchial epithelial BEAS‐2B cells, as well as in different primary cell types, including bronchial epithelial (HBE), vascular endothelial, fibroblast, and smooth muscle (ASM) cells, that are all present in the airways. Additionally, multiple GCs, including hydrocortisone, dexamethasone, and budesonide all induced KLF9 to a similar extent in A549 cells. This induction was abolished in A549 cells either by pretreating cells with the GR antagonist, Org34517, or by knocking down GR with siRNA. Chromatin immunoprecipitation of GR following glucocorticoid treatment in A549 and BEAS‐2B cells showed GR binding to sites 5.9, 6.7, 25, and 65 kb upstream of the KLF9 gene. Public domain data suggests that these sites may all possess enhancer activity and eRNA (enhancer RNA) expression was induced following GC treatment. Cellular fractionation showed that both basal and GC‐induced KLF9 protein was exclusively nuclear.CONCLUSIONSThe current work confirms that KLF9 is GC‐induced in multiple structural cell types (epithelial, fibroblast, airway smooth muscle and endothelial) that are found in the airways. In A549 cells the GC‐mediated induction of KLF9 was GR‐dependent. Since GR bound to, and appeared to activate, multiple enhancers upstream of the KLF9 gene, direct transactivation by GR is likely. GC induction of KLF9 therefore represents a highly conserved transactivation event that can be used to model transcriptional control by GR. Further deciphering the molecular events that occur at the KLF9 upstream promotor following GC treatment will enhance our understanding of how GR drives gene expression.Support or Funding InformationThis work was supported by: RN grants: Canadian Institutes of Health Research (CIHR), and Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant; MMM studentships: NSERC Postgraduate Scholarship – Doctoral, Queen Elizabeth II Doctoral scholarship, and The Lung Association – Alberta & NWT studentship award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.