Regulation of DNA methylation signatures on NF-κB and STAT3 pathway genes and TET activity in cigarette smoke extract-challenged cells/COPD exacerbation model in vitro.

Abstract

Chronic obstructive pulmonary disease (COPD) is a global health problem. Currently, there is a lack of knowledge about the pathobiology of this disease and available therapies are ineffective. Cigarette smoking is the leading cause of COPD; however, not all smokers develop COPD. Exacerbations of COPD caused by microbes are common and detrimental. Approximately 20-50% of patient exacerbations are caused by bacterial colonization in the lower airways. It is generally accepted that epigenetic mechanisms, especially DNA methylation, play an important role during progression of COPD. Thus, we hypothesized that DNA methylation patterns vary significantly following smoke exposure and during exacerbations caused by bacterial infections. To test our hypothesis, we used an in vitro study model that mimics COPD exacerbations and performed extensive studies to understand the role of CpG promoter methylation of NF-κB and STAT3-mediated pathway genes. Both NF-κB and STAT3 transcription factors play critical roles in orchestrating inflammatory responses during cigarette smoke exposure. In brief, human lung adenocarcinoma cells with type II alveolar epithelium characteristics (A549) were challenged with cigarette smoke extract (CSE) or DMSO (control) followed by a 3-h challenge with bacterial lipopolysaccharide (LPS; from Pseudomonas aeruginosa) prior to the termination of CSE exposure (COPD exacerbation group). The production of cytokines/chemokines, regulation of transcription factors, and DNA methylation of specific genes were then assessed. We also studied changes in the expression and activity of ten-eleven translocases (TETs), theenzymes responsible for DNA demethylation, and assessed their role in regulating DNA methylation in the CSE-challenged group. There was a significant increase in the release of cytokines/chemokines (IL-8, MCP-1, IL-6 and CCL5) in the COPD exacerbation group as compared to the control group. Hypomethylation of NF-κB-mediated pathway genes correlated with their induction in our COPD exacerbation study model. Further, we observed an important role of TET1/2 in regulating the DNA methylation of NF-κB, STAT3, IKK, and NIK genes and cytokine/chemokine production by A549 cells during CSE challenge. Studies to further define the role of TETs in CSE-mediated epigenetic regulation may lead to the development of better and more effective therapeutic intervention strategies for COPD.