Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1β (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6–24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B- and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B- and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokine-induced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3.

Rationale Clinically, mild‐to‐moderate asthma is well‐controlled by inhaled glucocorticoids. However, severe asthma is poorly controlled by this key pharmacological intervention. Understanding which pro‐inflammatory genes and signalling pathways avoid repression by glucocorticoids represents an important step towards improving current pharmacological interventions. IκB‐kinase‐ε (IKKε) was shown to escape full repression by glucocorticoids in airway epithelial cells, the primary site of action for glucocorticoid therapies in asthma. IKKε is also highly homologous to the canonical IκB kinases known to activate a family of pro‐inflammatory transcription factors: nuclear factor‐κB (NF‐κB). However, the role and regulation of IKKε is largely uncharacterized. This study aimed to elucidate the regulation of IKKε gene expression by NF‐κB in airway epithelial cells. Methods Human pulmonary epithelial A549 or bronchial airway epithelial BEAS‐2B cells were used to model gene expression in airway epithelial cells. Cells were treated with cytokines interleukin‐1β (IL1B, 1 ng/ml), tumour necrosis factor‐α (TNF, 10 ng/ml), and/or glucocorticoid (dexamethasone, 1µM). Western blotting, qPCR, and RNA‐sequencing were performed to characterize protein and mRNA expression of IKKε. Cells harbouring an NF‐κB driven luciferase reporter construct were used to assess overall NF‐κB activity. Chromatin immunoprecipitation (ChIP) sequencing data from BEAS‐2B cells was used to explore binding of NF‐κB subunit RelA/p65 to DNA using the UCSC genome browser. Results In A549 cells, IKKε protein was upregulated by both and IL1B and TNF with highest expression observed at 24h post stimulus. This peak expression was significantly, albeit partially, repressed by the glucocorticoid, dexamethasone. IKKε mRNA expression peaked at 6h, with partial repression by dexamethasone observed at 24h. ChIP‐seq data from BEAS‐2B cells treated with TNF showed p65 binding to the promoter region of IKKε. This suggests regulation of IKKε expression by NF‐κB. In A549 cells, silencing of p65 decreased the ability of IL1B to induce NF‐κB reporter activity and also reduced IL1B‐induced expression of IKKε. Likewise, adenoviral‐mediated overexpression of IκBαΔN, a dominant inhibitor of NF‐κB, markedly reduced IKKε mRNA expression that was induced by IL1B. Conclusions These data suggest that induction of IKKε expression by IL1B or TNF is dependent on NF‐κB activity in pulmonary epithelial cells. This supports the conclusion that early onset NF‐κB activity leads to the expression of inflammatory genes, such as IKKε, to activate later onset signal transduction cascades to prolong inflammatory gene expression in response to a given stimuli. As IKKε induction was only partially repressed by glucocorticoid, the induction of downstream effector processes may also be largely resistant to the repressive effects of glucocorticoid. These findings represent an important step in characterizing the role of IKKε in inflammatory signalling cascades and paves the way for interventions in sev...

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