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Differential BET protein recruitment to IL-6 and IL-8 promoters and suppression of innate immunity and cell cycle genes by BET inhibitors in TNFalpha/IL-17 stimulated human keratinocytes

ABSTRACT: Background: Epigenetic mechanisms are implicated in chronic inflammatory pathogenesis. Bromodomain Extra-Terminal (BET) proteins are druggable epi-reader proteins which bind acetylated histones and transcription factors regulating gene expression, including inflammatory genes. Objectives: To evaluate the role of specific histone acetylation events and BET protein interactions in promoting keratinocyte inflammatory and transcriptional responses, to determine the effects of pharmacological inhibition of BET proteins and thereby test the hypothesis that BET proteins represent an effective therapeutic target to treat cutaneous inflammation. Methods: Primary human keratinocytes were stimulated with tumour necrosis factor alpha and interleukin 17 (TNFalpha + IL-17), +/- BET inhibitor (I-BET151). Cytokine induced interleukin 6 and 8 (IL-6 /-8) responses were determined by qPCR and ELISA. Chromatin immunoprecipitation (ChIP) assays were undertaken to characterise epigenetic changes at the IL-6 and IL-8 gene promoters, while the effect of I-BET151 treatment on keratinocyte transcriptional response was determined by Illumina array. Results: TNFalpha + IL-17 induced gene-specific epigenetic changes, including: histone hyperacetylation, recruitment of BET proteins (BRD2/3/4) to the promoter regions of IL-6/-8 and activation of RNA polymerase II (PolII(S2P)), correlating with increased IL-6/-8 expression. I-BET151 reduced recruitment of BRD4 and activation of PolII(S2P), at the IL-6 promoter, and BRD3/4 and PolII(S2P), at the IL-8 promoter; which is consistent with I-BET151 inhibitory effects on TNFalpha + IL-17-induced IL-6/-8 expression. A ~10-fold enrichment of BRD4/p65 was observed at the IL-6 promoter compared to the IL-8 promoter. Transcriptomics analysis showed that I-BET151 modulated expression of genes involved in cell cycle, cell proliferation and the innate immune response in keratinocytes stimulated with TNFalpha + IL-17. Conclusions: In human keratinocytes, disease-relevant stimuli induced dynamic epigenetic changes at inflammatory gene loci. BET proteins played a critical role in keratinocyte innate immune responses and displayed differential effects; for example BRD4/p65 was particularly enriched at the IL-6 promoter, indicating that BRD4 may be a critical factor in integrating inflammatory stimuli via NF-B in chronic skin inflammation. Inhibition of these responses by I-BET151 highlights BET proteins as potential therapeutic targets in inflammatory skin disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155484 | GEO | 2020/12/31

REPOSITORIES: GEO

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Project description:Selective bromodomains inhibitors block the interaction between diverse bromodomains and extraterminal domains (BET) proteins and acetylated proteins. These inhibitors have shown beneficial effects in cancers malignancies and experimental inflammation in mouse models, but data on renal diseases are scarce. We have investigated the effect of the BET proteins inhibitor JQ1 in a mice model of unilateral ureteral obstruction. Treatment with JQ1 diminished renal damage, the presence of inflammatory cell infiltration and the upregulation of proinflammatory genes. The in vitro evaluation of JQ1 on TNF-Î± inducible genes in renal cells showed that BET inhibition modulated several biological processes, including inflammation or immune response. Moreover, gene-silencing experiments showed that BRD4 regulates several proinflammatory genes (IL-6, CCL-2 and CCL-5) and chromatin immunoprecipitation techniques demonstrated that BRD4 specifically binds to acetylated histone H3 in the promoter region of those genes. The nuclear factor-ï«B (NF-ï«B) pathway regulates renal inflammation. The RelA NF-ï«B subunit is activated by acetylation of lysine 310. In damaged kidneys and in TNF-Î±-treated renal cells, JQ1 blocked the nuclear translocation of RelA/NF-ï«B and NF-ï«B-mediated gene expression. Additionally, obstructed kidneys showed an activation of the Th17 immune response, which was diminished by JQ1 treatment. Our results demonstrate that the BET inhibition decreases renal inflammation by 3 independent mechanisms: 1) chromatin remodelling in the promoter regions of specific genes, 2) blocking NF-ï«B pathway activation, and 3) modulating the Th17 immune response. These results suggest that BET inhibitors could have important therapeutic applications in inflammatory renal diseases. HK2 cell line was treated with JQ1 or its enantiomer JQ1(-) (500nM) for 1 hour before stimulation or not with TNF-Î± (5ng/ml) for additional 3h. We analized the genes up- and down-regulated by TNF-Î± vs unstimulated cells, and further the genes downregulated or unaffected by JQ1 compared to the cells treated with the enantiomer (-) JQ1.

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Differential BET protein recruitment to IL-6 and IL-8 promoters and suppression of innate immunity and cell cycle genes by BET inhibitors in TNFalpha/IL-17 stimulated human keratinocytes

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