Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015755
ISSN1083-351X
AutoresMahmoud Mostafa, Akanksha Bansal, Aubrey N. Michi, Sarah K. Sasse, David Proud, Anthony N. Gerber, Robert Newton,
Tópico(s)Genomics and Chromatin Dynamics
ResumoLigand-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. 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. 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Airway epithelial cells are not only critical in the pathogenesis of asthma, but also represent key effector cells in mediating therapeutic responses to glucocorticoids in dampening airway and lung inflammation (39Lambrecht B.N. Hammad H. The airway epithelium in asthma.Nat. Med. 2012; 18: 684-692Crossref PubMed Scopus (557) Google Scholar, 40Klaßen C. Karabinskaya A. Dejager L. Vettorazzi S. Van Moorleghem J. Lühder F. Meijsing S.H. Tuckermann J.P. Bohnenberger H. Libert C. Reichardt H.M. Airway epithelial cells are crucial targets of glucocorticoids in a mouse model of allergic asthma.J. Immunol. 2017; 199: 48-61Crossref PubMed Scopus (24) Google Scholar). The current study therefore takes advantage of the established "in common" glucocorticoid regulation of KLF9 in primary human bronchial epithelial (HBE) cells and both alveolar type II (A549) and bronchial (BEAS-2B) epithelial cell lines (8Mostafa M.M. Rider C.F. Shah S. Traves S.L. Gordon P.M.K. Miller-Larsson A. Leigh R. Newton R. Glucocorticoid-driven transcriptomes in human airway epithelial cells: commonalities, differences and functional insight from cell lines and primary cells.BMC Med. Genomics. 2019; 12: 29Crossref PubMed Scopus (9) Google Scholar, 14Leigh R. Mostafa M.M. King E.M. Rider C.F. Shah S. Dumonceaux C. Traves S.L. McWhae A. Kolisnik T. Kooi C. Slater D.M. Kelly M.M. Bieda M. Miller-Larsson A. Newton R. An inhaled dose of budesonide induces genes involved in transcription and signaling in the human airways: enhancement of anti- and proinflammatory effector genes.Pharmacol. Res. Perspect. 2016; 4e00243Crossref PubMed Scopus (30) Google Scholar). These cell lines represent well-studied human models, for which high-quality genomic data exists (5Reddy T.E. Pauli F. Sprouse R.O. Neff N.F. Newberry K.M. Garabedian M.J. Myers R.M. Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation.Genome Res. 2009; 19: 2163-2171Crossref PubMed Scopus (347) Google Scholar, 19Kadiyala V. Sasse S.K. Altonsy M.O. Berman R. Chu H.W. Phang T.L. Gerber A.N. Cistrome-based cooperation between airway epithelial glucocorticoid receptor and NF-κB orchestrates anti-inflammatory effects.J. Biol. Chem. 2016; 291: 12673-12687Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 20Sasse S.K. Gruca M. Allen M.A. Kadiyala V. Song T. Gally F. Gupta A. Pufall M.A. Dowell R.D. Gerber A.N. Nascent transcript analysis of glucocorticoid crosstalk with TNF defines primary and cooperative inflammatory repression.Genome Res. 2019; 29: 1753-1765Crossref PubMed Scopus (10) Google Scholar, 24McDowell I.C. Barrera A. D'Ippolito A.M. Vockley C.M. Hong L.K. Leichter S.M. Bartelt L.C. Majoros W.H. Song L. Safi A. Koçak D.D. Gersbach C.A. Hartemink A.J. Crawford G.E. Engelhardt B.E. et al.Glucocorticoid receptor recruits to enhancers and drives activation by motif-directed binding.Genome Res. 2018; 28: 1272-1284Crossref PubMed Scopus (33) Google Scholar, 26Vockley C.M. D'Ippolito A.M. McDowell I.C. Majoros W.H. Safi A. Song L. Crawford G.E. Reddy T.E. Direct GR binding sites potentiate clusters of TF binding across the human genome.Cell. 2016; 166: 1269-1281.e19Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 27D'Ippolito A.M. McDowell I.C. Barrera A. Hong L.K. Leichter S.M. Bartelt L.C. Vockley C.M. Majoros W.H. Safi A. Song L. Gersbach C.A. Crawford G.E. Reddy T.E. Pre-established chromatin interactions mediate the genomic response to glucocorticoids.Cell Syst. 2018; 7: 1-15PubMed Google Scholar), and are here used to explore genomic features that may impact on the GR-mediated expression of KLF9 in therapeutically relevant cells. Expression data for all 17 KLF genes were extracted from previously published transcriptome analyses of A549, BEAS-2B, and primary HBE cells, each following no stimulation or treatment with maximally effective concentrations of the synthetic glucocorticoid budesonide for 6 h (8Mostafa M.M. Rider C.F. Shah S. Traves S.L. Gordon P.M.K. Miller-Larsson A. Leigh R. Newton R. Glucocorticoid-driven transcriptomes in human airway epithelial cells: commonalities, differences and functional insight from cell lines and primary cells.BMC Med. Genomics. 2019; 12: 29Crossref PubMed Scopus (9) Google Scholar). These data were compared with the effect of an inhaled dose (1200 μg) of budesonide in human bronchial biopsies taken 6 h post placebo or budesonide inhalation (14Leigh R. Mostafa M.M. King E.M. Rider C.F. Shah S. Dumonceaux C. Traves S.L. McWhae A. Kolisnik T. Kooi C. Slater D.M. Kelly M.M. Bieda M. Miller-Larsson A. Newton R. An inhaled dose of budesonide induces genes involved in transcription and signaling in the human airways: enhancement of anti- and proinflammatory effector genes.Pharmacol. Res. Perspect. 2016; 4e00243Crossref PubMed Scopus (30) Google Scholar). While various KLFs, including KLF15, revealed modulation by budesonide, only KLF9 showed significant (p ≤ 0.05), ≥2-fold, induction by budesonide in all three epithelial cell variants and in vivo in airway biopsy tissues (Fig. 1A). In addition, comparable increases in KLF9 mRNA expression in response to budesonide were observed in primary human airway smooth muscle (ASM), bronchial fibroblast (HBF), and vascular endothelial (HUVEC) cells (Fig. S1A). Thus, not only was the induction of 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. In A549 cells treated with budesonide for 1, 2, 6, 12, and 24 h, KLF4 mRNA was modestly (∼2-fold) induced at 1 and 2 h, whereas KLF6 and, to a greater extent, KLF9 were more substantially (≥2-fold) and significantly induced at all times (Fig. 1B). Western blotting showed significant increases in KLF9 protein at 2 h and a plateau of peak expression that extended from 4 to 24 h (Fig. 1C). Similarly, transcriptomic data from BEAS-2B cells revealed significant (p ≤ 0.05) ≥2-fold induction of KLF4, KLF6, and KLF7 mRNAs by budesonide with peak expression occurring at 1 or 2 h (Fig. S1B) (8Mostafa M.M. Rider C.F. Shah S. Traves S.L. Gordon P.M.K. Miller-Larsson A. Leigh R. Newton R. Glucocorticoid-driven transcriptomes in human airway epithelial cells: commonalities, differences and functional insight from cell lines and primary cells.BMC Med. Genomics. 2019; 12: 29Crossref PubMed Scopus (9) Google Scholar). KLF15 mRNA also showed significant, ≥2-fold budesonide-induced expression that peaked at 6 h. KLF9 mRNA revealed peak expression at 2 h, but was significantly induced, ≥2-fold, at all times (1, 2, 6, and 18 h) analyzed. Western blotting indicated increased KLF9 protein from 1 h post budesonide treatment of BEAS-2B cells that peaked at 6 h and remained highly elevated at 18 h (Fig. S1C). Several studies have reported KLF9 regulation by various nuclear hormone receptor (NHR) agonists, including thyroxine, progesterone, and estrogen (reviewed in (41Knoedler J.R. Denver R.J. Krüppel-like factors are effectors of nuclear receptor signaling.Gen. Comp. Endocrinol. 2014; 203: 49-59Crossref PubMed Scopus (33) Google Scholar)). To assess their influence on KLF9 expression, A549 cells were treated with agonists of the glucocorticoid (GR), progesterone (PGR), estrogen (ESR), thyroxine (THRs), vitamin D (VDR), retinoic acid (RARs), retinoid (RXRs), RAR-related (RORs), or Rev-Erb (NR1Ds) receptors (Fig. S1D). Budesonide and triiodo-L-thyronine, agonists at GR and THRs, induced KLF9 mRNA expression by 6.9 and 2.8-fold, respectively (both p ≤ 0.05). Dihydroxyvitamin D3, a VDR agonist, also induced KLF9 expression by 2.2-fold, but this effect was variable and did not reach significance. Whereas other NHR ligands were largely without effect, the above data indicate glucocorticoids, presumably acting through GR, to be a major regulator of KLF9 expression in the lungs, including the epithelium, as modeled by A549 cells. While glucocorticoids are used for their anti-inflammatory effects, a significant fraction of the glucocorticoid-regulated transcriptome is modulated by inflammatory stimuli, including lipopolysaccharide (LPS) and TNF (20Sasse S.K. Gruca M. Allen M.A. Kadiyala V. Song T. Gally F. Gupta A. Pufall M.A. Dowell R.D. Gerber A.N. Nascent transcript analysis of glucocorticoid crosstalk with TNF defines primary and cooperative inflammatory repression.Genome Res. 2019; 29: 1753-1765Crossref PubMed Scopus (10) Google Scholar, 33Chinenov Y. Coppo M. Gupte R. Sacta M.A. Rogatsky I. Glucocorticoid receptor coordinates transcription facto
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