Abstract
Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air–liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5ʹ-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.
Highlights
Glucocorticoids are adrenal hormones that play crucial roles in metabolic homeostasis, development, responses to stress, and inflammation [1, 2]
Expression data for all 17 Krüppel-like factor (KLF) genes were extracted from previously published transcriptome analyses of A549, BEAS2B, and primary human bronchial epithelial (HBE) cells, each following no stimulation or treatment with maximally effective concentrations of the synthetic glucocorticoid budesonide for 6 h [8]
Was the induction of Krüppel-like factor 9 (KLF9) mRNA expression by budesonide a conserved response in the airways in vivo, as well as in structural cells relevant to the airways, but the effect was recapitulated in the model epithelial cell lines, A549 and BEAS-2B
Summary
Glucocorticoids are adrenal hormones that play crucial roles in metabolic homeostasis, development, responses to stress, and inflammation [1, 2]. Similar to KLF9 usRNA, eRNAs from the four regions tested were significantly induced by glucocorticoid at the earliest time points in both A549 and HBE cells (Fig. 3, C–D).
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