Brd4 is essential for IL-1β-induced inflammation in human airway epithelial cells.
BACKGROUND
Chronic inflammation and oxidative stress are key features of chronic obstructive pulmonary disease (COPD). Oxidative stress enhances COPD inflammation under the control of the pro-inflammatory redox-sensitive transcription factor nuclear factor-kappaB (NF-κB). Histone acetylation plays a critical role in chronic inflammation and bromodomain and extra terminal (BET) proteins act as "readers" of acetylated histones. Therefore, we examined the role of BET proteins in particular Brd2 and Brd4 and their inhibitors (JQ1 and PFI-1) in oxidative stress- enhanced inflammation in human bronchial epithelial cells.


METHODS
Human primary epithelial (NHBE) cells and BEAS-2B cell lines were stimulated with IL-1β (inflammatory stimulus) in the presence or absence of H2O2 (oxidative stress) and the effect of pre-treatment with bromodomain inhibitors (JQ1 and PFI-1) was investigated. Pro-inflammatory mediators (CXCL8 and IL-6) were measured by ELISA and transcripts by RT-PCR. H3 and H4 acetylation and recruitment of p65 and Brd4 to the native IL-8 and IL-6 promoters was investigated using chromatin immunoprecipitation (ChIP). The impact of Brd2 and Brd4 siRNA knockdown on inflammatory mediators was also investigated.


RESULTS
H2O2 enhanced IL1β-induced IL-6 and CXCL8 expression in NHBE and BEAS-2B cells whereas H2O2 alone did not have any affect. H3 acetylation at the IL-6 and IL-8 promoters was associated with recruitment of p65 and Brd4 proteins. Although p65 acetylation was increased this was not directly targeted by Brd4. The BET inhibitors JQ1 and PFI-1 significantly reduced IL-6 and CXCL8 expression whereas no effect was seen with the inactive enantiomer JQ1(-). Brd4, but not Brd2, knockdown markedly reduced IL-6 and CXCL8 release. JQ1 also inhibited p65 and Brd4 recruitment to the IL-6 and IL-8 promoters.


CONCLUSIONS
Oxidative stress enhanced IL1β-induced IL-6 and CXCL8 expression was significantly reduced by Brd4 inhibition. Brd4 plays an important role in the regulation of inflammatory genes and provides a potential novel anti-inflammatory target.
Airways Disease Section, National Heart & Lung Institute, Imperial College London, London, United Kingdom.
Younis M
YM
Khan
School of Applied Sciences, University of Wolverhampton, Wolverhampton, United Kingdom.
Paul
P
Kirkham
Airways Disease Section, National Heart & Lung Institute, Imperial College London, London, United Kingdom.
Peter J
PJ
Barnes
Airways Disease Section, National Heart & Lung Institute, Imperial College London, London, United Kingdom.
Ian M
IM
Adcock
D001381:Azepines / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007249:Inflammation / Q000139:chemically induced* / Q000378:metabolism*; D053583:Interleukin-1beta / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014230:Triazoles / Q000494:pharmacology; D000092468:Bromodomain Containing Proteins
D001381:Azepines; D018797:Cell Cycle Proteins; D006861:Hydrogen Peroxide; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D014157:Transcription Factors; D014230:Triazoles; C561695:(+)-JQ1 compound; C098415:BRD2 protein, human; D000092468:Bromodomain Containing Proteins; C441289:BRD4 protein, human