Epigenetic modifying enzyme expression in asthmatic airway epithelial cells and fibroblasts.

BACKGROUND: Recognition of the airway epithelium as a central mediator in the pathogenesis of asthma has necessitated greater understanding of the aberrant cellular mechanisms of the epithelium in asthma. The architecture of chromatin is integral to the regulation of gene expression and is determined by modifications to the surrounding histones and DNA. The acetylation, methylation, phosphorylation, and ubiquitination of histone tail residues has the potential to greatly alter the accessibility of DNA to the cells transcriptional machinery. DNA methylation can also interrupt binding of transcription factors and recruit chromatin remodelers resulting in general gene silencing. Although previous studies have found numerous irregularities in the expression of genes involved in asthma, the contribution of epigenetic regulation of these genes is less well known. We propose that the gene expression of epigenetic modifying enzymes is cell-specific and influenced by asthma status in tissues derived from the airways. METHODS: Airway epithelial cells (AECs) isolated by pronase digestion or endobronchial brushings and airway fibroblasts obtained by outgrowth technique from healthy and asthmatic donors were maintained in monolayer culture. RNA was analyzed for the expression of 82 epigenetic enzymes across 5 families of epigenetic modifying enzymes. Western blot and immunohistochemistry were also used to examine expression of 3 genes. RESULTS: Between AECs and airway fibroblasts, we identified cell-specific gene expression in each of the families of epigenetic modifying enzymes; specifically 24 of the 82 genes analyzed showed differential expression. We found that 6 histone modifiers in AECs and one in fibroblasts were differentially expressed in cells from asthmatic compared to healthy donors however, not all passed correction. In addition, we identified a corresponding increase in Aurora Kinase A (AURKA) protein expression in epithelial cells from asthmatics compared to those from non-asthmatics. CONCLUSIONS: In summary, we have identified cell-specific variation in gene expression in each of the families of epigenetic modifying enzymes in airway epithelial cells and airway fibroblasts. These data provide insight into the cell-specific variation in epigenetic regulation which may be relevant to cell fate and function, and disease susceptibility.

Action of 1,25(OH)2D3 on Human Asthmatic Bronchial Fibroblasts: Implications for Airway Remodeling in Asthma.

BACKGROUND: Airway fibroblasts are major contributors to the histopathological feature of airway remodeling in asthma by their implication in the cell invasiveness and profibrogenic secretory phenotype observed in subepithelial fibrosis. 1,25 Dihydroxy vitamin D3 (1,25(OH)2D3) is an important therapeutic agent that blocks many features of airway remodeling induced by profibrogenic mediators, such as transforming growth factor beta 1 (TGF-ß1) or T helper type 1 inflammatory cytokines. OBJECTIVE: We hypothesized that 1,25(OH)2D3 opposes the TGF-ß1 or tumor necrosis factor alpha (TNF-α)-Interleukin 1 beta (IL-1ß) stimulation on airway fibroblast profibrogenic secretory phenotype observed in severe asthmatic patients. Our aim was to investigate the anti-fibrogenic effect of 1,25(OH)2D3 in TGF-ß1 or TNF-α-IL-1ß-stimulated human bronchial fibroblast cells (HBFCs) from severe asthmatic compared with non-asthmatic subjects. PATIENTS AND METHODS: All experiments were performed on primary HBFCs from asthmatic (DHBFCs, n=4) and non-asthmatic subjects (NHBFCs, n=4). mRNA expression and protein quantification of key fibrogenic markers were analyzed by RT-qPCR and ELISA, comparing HBFCs from asthmatic and non-asthmatic subjects. Vitamin D receptor (VDR) mRNA expression and its functionality in HBFCs were assessed by RT-qPCR. HBFCs proliferation was assessed by flow cytometry using BrdU-FITC/7AAD bivariate staining, while HBFCs apoptosis by Annexin V-FITC/7AAD. RESULTS: VDR is constitutively expressed in HBFCs and the addition of 1,25(OH)2D3 significantly increased mRNA expression of CYP24A1 (a direct VDRs' target gene) in both HBFCs groups. DHBFCs cultured in the presence of TGF-ß1 or TNF-α-IL-1ß showed increased mRNA expression and protein secretion of fibrogenic markers when compared to NHBFCs. Additionally, we observed decreased mRNA expression of FN 1, LUM, BGN, MMP2, COL5A1, TIMP1 and CC-chemokines (CCL2, CCL5, CCL11) in response to 1,25(OH)2D3 addition to the TGF-ß1 or TNF-α-IL-1ß-stimulated HBFCs. Cell culture media obtained from TGF-ß1 or TNF-α-IL-1ß-stimulated DHBFCs showed decreased protein secretion (fibronectin 1, lumican, MCP1, RANTES and eotaxin-1) in response to 1,25(OH)2D3 when compared to NHBFCs. 1,25(OH)2D3 inhibited proliferation in TGF-ß1-stimulated HBFCs through G0/G1 cell cycle arrest and these effects were not correlated with the induction of apoptosis. CONCLUSION: DHBFCs under TGF-ß1 or TNF-α-IL-1ß stimulation showed higher fibrogenic capacity when compared to NHBFCs. 1,25(OH)2D3 significantly blocked these effects and highlight 1,25(OH)2D3 as a possible therapeutic target for severe asthma.