Inducible gene expression of IκB-kinase ε is dependent on nuclear factor-κB in human pulmonary epithelial cells

• Published in: Biochemical journal Vol. 481; no. 14; p. 959
• Main Authors: Necker-Brown, Amandah, Kooi, Cora, Thorne, Andrew J, Bansal, Akanksha, Mostafa, Mahmoud M, Chandramohan, Priyanka, Gao, Alex, Kalyanaraman, Keerthana, Milani, Arya, Gill, Sachman, Georgescu, Andrei, Sasse, Sarah K, Gerber, Anthony N, Leigh, Richard, Newton, Robert
• Format: Journal Article
• Language: English
• Published: England 17-07-2024
• Subjects: A549 Cells; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Gene Expression Regulation - drug effects; Humans; I-kappa B Kinase - genetics; I-kappa B Kinase - metabolism; Interleukin-1beta - genetics; Interleukin-1beta - metabolism; Interleukin-1beta - pharmacology; Lung - cytology; Lung - metabolism; NF-kappa B - genetics; NF-kappa B - metabolism; Respiratory Mucosa - cytology; Respiratory Mucosa - metabolism; Transcription Factor RelA - genetics; Transcription Factor RelA - metabolism; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - metabolism; Tumor Necrosis Factor-alpha - pharmacology; epithelial cells; nuclear factor κB; promoter regions; IκB kinase
• DOI: 10.1042/BCJ20230461
• EISSN: 1470-8728

While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1β and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1β- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1β- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1β may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.