ABSTRACT
Airway epithelial cells in cystic fibrosis (CF) overexpress Interleukin 8 (CXCL8) through poorly defined mechanisms. CXCL8 transcription is dependent on coordinated binding of CCAAT/enhancer binding protein (C/EBP)ß, nuclear factor (NF)-κB, and activator protein (AP)-1 to the promoter. Here we show abnormal epigenetic regulation is responsible for CXCL8 overexpression in CF cells. Under basal conditions CF cells had increased bromodomain (Brd)3 and Brd4 recruitment and enhanced NF-κB and C/EBPß binding to the CXCL8 promoter compared to non-CF cells due to trimethylation of histone H3 at lysine 4 (H3K4me3) and DNA hypomethylation at CpG6. IL-1ß increased NF-κB, C/EBPß and Brd4 binding. Furthermore, inhibitors of bromodomain and extra-terminal domain family (BET) proteins reduced CXCL8 production in CF cells suggesting a therapeutic target for the BET pathway.
Subject(s)
Cystic Fibrosis/genetics , Epigenesis, Genetic , Interleukin-8/genetics , Azepines/pharmacology , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Cycle Proteins , Cells, Cultured , Cystic Fibrosis/pathology , DNA Methylation , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Interleukin-1beta/pharmacology , Interleukin-8/metabolism , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Triazoles/pharmacologyABSTRACT
Asthma is characterized by airway inflammation and remodeling and CXCL8 is a CXC chemokine that drives steroid-resistant neutrophilic airway inflammation. We have shown that airway smooth muscle (ASM) cells isolated from asthmatic individuals secrete more CXCL8 than cells from nonasthmatic individuals. Here we investigated chromatin modifications at the CXCL8 promoter in ASM cells from nonasthmatic and asthmatic donors to further understand how CXCL8 is dysregulated in asthma. ASM cells from asthmatic donors had increased histone H3 acetylation, specifically histone H3K18 acetylation, and increased binding of histone acetyltransferase p300 compared with nonasthmatic donors but no differences in CXCL8 DNA methylation. The acetylation reader proteins Brd3 and Brd4 were bound to the CXCL8 promoter and Brd inhibitors inhibited CXCL8 secretion from ASM cells by disrupting Brd4 and RNA polymerase II binding to the CXCL8 promoter. Our results show a novel dysregulation of CXCL8 transcriptional regulation in asthma characterized by a promoter complex that is abnormal in ASM cells isolated from asthmatic donors and can be modulated by Brd inhibitors. Brd inhibitors may provide a new therapeutic strategy for steroid-resistant inflammation.
Subject(s)
Asthma/metabolism , Interleukin-8/metabolism , Muscle, Smooth/metabolism , Myocytes, Smooth Muscle/metabolism , Nerve Tissue Proteins/metabolism , Acetylation , Airway Remodeling/immunology , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Cycle Proteins , Cells, Cultured , DNA Methylation , DNA-Binding Proteins/metabolism , Histones/metabolism , Humans , Inflammation/immunology , Interleukin-8/antagonists & inhibitors , Interleukin-8/genetics , Nerve Tissue Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Primary Cell Culture , Promoter Regions, Genetic , Protein Binding , RNA Polymerase II/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , Transcription Factor RelA/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transcription, Genetic , p300-CBP Transcription Factors/metabolismABSTRACT
Monocyte chemotactic protein-1 (MCP-1) is a member of the CC family of cytokines. It has monocyte and lymphocyte chemotactic activity and stimulates histamine release from basophils. MCP-1 is implicated in the pathogenesis of inflammatory diseases, including asthma. The airway smooth muscle (ASM) layer is thickened in asthma, and the growth factors and cytokines secreted by ASM cells play a role in the inflammatory response of the bronchial wall. Glucocorticoids and ß(2)-agonists are first-line drug treatments for asthma. Little is known about the effect of asthma treatments on MCP-1 production from human ASM cells. Here, we determined the effect of ciclesonide (a glucocorticoid) and formoterol (a ß(2)-agonist) on MCP-1 production from human ASM cells. TNFα and IL-1ß induced MCP-1 secretion from human ASM cells. Formoterol had no effect on MCP-1 expression, while ciclesonide significantly inhibited IL-1ß- and TNFα-induced MCP-1. Furthermore, ciclesonide inhibited IL-1ß- and TNFα-induced MCP-1 mRNA and IL-1ß- and TNFα-induced MCP-1 promoter and enhancer luciferase reporters. Western blots showed that ciclesonide had no effect on IκB degradation. Finally, ciclesonide inhibited an NF-κB luciferase reporter. Our data show that ciclesonide inhibits IL-1ß- and TNFα-induced MCP-1 production from human ASM cells via a transcriptional mechanism involving inhibition of NF-κB binding.
