IL-13 Augments Histone Demethylase JMJD2B/KDM4B Expression Levels, Activity, and Nuclear Translocation in Airway Fibroblasts in Asthma

Abstract

Purpose: Asthma is one of the most common obstructive pulmonary diseases worldwide. Epigenetic alterations, including DNA methylation and histone modifications, have been reported to contribute to asthma pathogenesis. Since the inflammation mediator and remodeling trigger, IL-13, is known to play a central role in the pathophysiology of asthma, this study was aimed to identify novel IL-13-regulated epigenetic modifiers in asthma that may contribute to subepithelial fibrosis.

Methods: Publicly available transcriptomic datasets from Gene Expression Omnibus (GEO) were used to identify differentially expressed genes on an epigenetic level upon IL-13 exposure in lung fibroblasts. Bronchial fibroblasts isolated from healthy and asthmatic individuals were assessed for the gene and protein expression levels of the identified gene at baseline and upon IL-13 treatment using qRT-PCR and western blotting, respectively. Its subcellular localization and tissue distribution were examined in bronchial fibroblasts as well as bronchial biopsies by immunofluorescence and immunohistochemical analysis, respectively.

Results: Bioinformatic analysis revealed the differential expression of the histone demethylase JMJD2B/KDM4B, a well-known epigenetic modulator that leads to the demethylation of different lysine residues on histones, in IL-13-treated lung fibroblasts. The baseline expression levels of JMJD2B were higher in asthmatic fibroblasts and in bronchial biopsies in comparison to healthy ones. There was also an increase in JMJD2B activity as evidenced by the demethylation of its downstream target, H3K36me3. Furthermore, IL-13 stimulation induced JMJD2B expression and further demethylation of H3K36me3 in asthmatic fibroblasts. This was accompanied by increased translocation of JMJD2B into the nucleus.

Conclusion: This study highlights the novel pathological involvement of the histone demethylase JMJD2B/KDM4B in asthmatic airway fibroblasts that are regulated by IL-13. Clinical implications. Given that there is no single therapeutic medicine to effectively treat the various subtypes of asthma, this study provides promising insights into JMJD2B as a new therapeutic target that could potentially improve the treatment and management of asthma.

Figure 1

Normalized gene expression of four DEGs genes (JMJD2C, JMJD2B, PTDSR, and JMJD3) was involved in histone trimethylation and or demethylation as extracted from GSE56338 dataset. Adult lung fibroblasts (passage 6) treated with IL-13 (non-il13) compared to the same cells treated with media only (non-non).

Figure 2

Baseline differential levels of histone demethylase JMJD2B and downstream targets in healthy and asthmatic fibroblasts. (a) qRT-PCR analysis of JMJD2B expression levels. (b) Western bolt analysis showing JMJD2B and trimethylated histones H3 lysine residues at K4, K9, K27, K36, and K79 protein levels in the fibroblasts. (b) Calculated mean ± SD fold change in protein-expression levels in normal and asthma fibroblasts based on two separate experiments. ^∗∗∗p < 0.01, determined using unpaired two-tailed Student t-test. Representative immunoblots depicting protein levels normal and asthma fibroblasts where β-actin was used as loading control.

Figure 3

JMJD2B and its downstream target levels after IL-13 stimulation in normal and asthmatic fibroblasts. (a) qRT-PCR analysis of JMJD2B expression levels upon IL-13 stimulation in normal and asthma fibroblasts. (b) qRT-PCR analysis of JMJD2B expression levels comparing fibroblasts with or without IL-13 stimulation in normal and asthma fibroblasts. (c) Western bolt analysis showing JMJD2B and trimethylated histones H3 lysine residue at K36 protein levels in the fibroblasts upon IL-13 stimulation. (d) Calculated mean ± SD fold change in protein-expression levels comparing fibroblasts with or without IL-13 stimulation in normal and asthma fibroblasts based on two separate experiments. Graphical data are represented as mean ± SEM. ^∗∗∗p < 0.01, determined using unpaired two-tailed Student t-test. Representative immunoblots depicting protein levels normal and asthma fibroblasts where β-actin was used as loading control.

Figure 4

JMJD2B subcellular localization after IL-13 stimulation in normal and asthmatic fibroblasts. Immunofluorescence staining of in normal and asthmatic fibroblasts stimulated with IL-13 that were stained for DNA (DAPI; blue), JMJD2B (green), β-actin (red). The images were observed under a microscope at 20x magnification (a) and at 40x magnification (b). Arrows indicate subcellular localization of JMJD2B in fibroblasts.

Figure 5

JMJD2B expression in asthmatic bronchial biopsy tissues. Representative bronchial biopsy sections from healthy control showing weak and asthmatic showing moderate to strong JMJD2B protein expression. Representative images for H&E staining taken at 10x magnification and IHC for JMJD2B taken at 20x magnification showing developed with 3,3′- diaminobenzidine (brown). Nuclei were counterstained with hematoxylin (blue). Arrows refer to bronchial epithelium. Arrowheads refer to fibroblasts.