Cigarette smoking is a critical risk factor for the development and progression of chronic obstructive pulmonary disease (COPD), characterized by chronic bronchitis and emphysema (1). Cigarette smoke (CS) induces inflammatory responses and oxidative stress by altering the cellular redox status, resulting in epigenetic modifications. Several studies have demonstrated that epigenetic enzymes including DNA methyl-transferase, histone (de)acetylase, and histone (de)methylase, act as crucial regulators of COPD pathogenesis (2). For example, suppression of Histone deacetylase 2 (HDAC2) increases pro-inflammatory cytokine production through NF-κB signaling in CS-exposed cells and patients with COPD (3). Histone deacetylase SIRT1-regulated expression of matrix metalloproteinase 9 is associated with inflammation and lung remodeling in patients with COPD (4). Although epigenetic modifiers are widely reported to be involved in the regulation of COPD pathogenesis (5), the role of histone demethylases in this phenomenon is not well studied. Recent studies revealed that the expression of USP38, a potential deubiquitylating enzyme of lysine-specific demethylase 1 (LSD1), also known as KDM1A, is upregulated in the COPD lung tissue. USP38 enhances the stability of LSD1, thereby activating signaling pathways and promoting cell proliferation and colony formation (6, 7), which suggests a role of LSD1 in COPD pathogenesis.
LSD1 is a member of the flavin adenine dinucleotide-dependent amine oxidase family (8). LSD1 was first isolated as an interaction partner of the histone deacetylase HDAC2 and identified as a histone demethylase (9). LSD1 specifically removes methyl groups from histone H3 lysine 4 and H3 lysine 9, and thus, can operate as a transcriptional repressor or activator, respectively (10). Furthermore, LSD1 can demethylate and regulate the function and stability of non-histone proteins such as p53, DNMT1, HIF-1α and p65, which are involved in gene expression (11). Recent evidence showed that several kinases, such as PKCα, PLK1, CK2 and GSK3β, phosphorylate serine residues in LSD1 and thereby, regulate its biological functions (12-16). For example, PKCα-mediated phosphorylation of LSD1 on S112 regulates diverse biological processes including circadian rhythm, presynaptic plasticity, epithelial-mesenchymal transition, and inflammatory responses (12, 15, 17, 18). Furthermore, phosphorylation of LSD1 by PKCα represents a crucial mode of epigenetic regulation in inflammatory diseases, such as sepsis and inflammatory bowel disease in murine models (15, 19). However, little is known about the precise role of LSD1 in CS-induced pulmonary inflammation in the COPD model.
In this study, we explored the effects of Lsd1-S112A on the inflammatory response and oxidative stress in the COPD model using phosphorylation defective mutant
To investigate the role of Lsd1-S112A in the onset of COPD in vivo, as an initial approach,
To address whether
Exposure to CSE promotes the pathogenesis of COPD by increasing oxidative stress and inflammation in the lung (22). To examine whether the phosphorylation status of LSD1 affects airway inflammation and oxidative stress following CSE and LPS exposure, we compared the expression of Cox-2 and Ho-1 in the
Although we detected elevated expression of Cox-2 and Ho-1 in the lungs of
To validate the hypothesis that the phosphorylation defect of Lsd1-S112 is critical for the expression of Cox-2 and Ho-1 proteins in MEFs, we used Go6976, a PKCα inhibitor, to block the phosphorylation of Lsd1-S112. Immunoblot analysis was performed on MEF extracts following 24 h CSE and LPS treatment in the absence or presence of Go6976. Interestingly, pre-treatment with Go6976 enhanced Cox-2 and Ho-1 expression (Fig. 4A, B). In a separate experiment, MEFs were treated with Go6976 or vehicle for 1 h and then treated with CSE, LPS, or CSE/LPS for 4 h to evaluate the
LSD1 is an epigenetic regulator that controls lysine methylation status of histones and non-histone proteins, and its biological functions are regulated by post-translational modifications including phosphorylation (12, 15, 17, 18). Emerging evidence indicates that phosphorylation of LSD1 at serine 112 (equi-valent to serine 111 on the human protein) plays a crucial role in the regulation of inflammatory responses in sepsis and IBD models (15, 19). To the best of our knowledge, this is the first study to demonstrate the effect of Lsd1-S112A on the murine CSE/LPS-induced COPD model.
Chronic inflammation and oxidative stress are important features of the pathogenesis of COPD. CS, a potent inducer of neutrophilic inflammation, alters the inflammatory responses by modulating the production of cytokines and chemokines with consecutive recruitment of macrophages and neutrophils, and thus, causing further damage to the lung tissue (25). CS is a major exogenous source of oxidative stress in the lung. Oxidative stress is implicated in airway inflammation and is one of the major prognostic factors in the pathogenesis of COPD. High levels of ROS are generated by neutrophils following CS exposure and contribute to oxidative stress (21). In the current study, we showed that intratracheal instillation of CSE combined with LPS led to increased immune cell influx and more severe parenchymal destruction in
Taken together, our findings reveal that the phosphorylation defect on Lsd1-S112 is critical for inflammation and oxidative stress upon CSE/LPS exposure and plays a key role in the exacerbation of COPD. This study provides new insights into the correlation between LSD1 phosphorylation and oxidative stress in exacerbating COPD.
We used THIS cigarettes (KT&G, Korea), and each cigarette contained 1.1 mg nicotine, 15 mg tar, and 15 mg carbon monoxide. Briefly, the smoke from five cigarettes was bubbled slowly through 100 ml phosphate-buffered saline using a vacuum pump, which was considered as a 100% CSE solution. The soluble CSE was then sterilized and stored at −80°C.
Mice were treated with CSE, LPS or CSE/LPS solutions for establishing the COPD murine model (Fig. 1). Briefly, the solutions were administered by intratracheal injection 4 times at 7-day intervals. The mice were divided into four groups as follows; control group (100 μl PBS), CSE group (100 μl CSE), LPS group (10 μg LPS in 100 μl PBS) and CSE/LPS group (10 μg LPS in 100 μl CSE). The mice were euthanized at day 7 or 24 h after the last dose.
BALF was obtained through the tracheal cannula by washing the airway lumen with 1 ml of sterile saline. Total cell counts in the BALF were determined using a hemocytometer after lysis of red blood cells with a red blood cell lysis buffer (Sigma, USA). Differential cell counts in the BALF were examined by Cytospin preparation followed by staining with the Diff-Quik reagent (Sysmex International Reagents, Kobe, Japan).
The left lung tissue of each mouse was fixed with an intratracheal injection of 4% paraformaldehyde and immersed in the same fixative to preserve pulmonary architecture. The fixed lungs were then embedded in paraffin using standard procedures. Lung sections (4 μm) were stained with H&E for histological examinations. MLI and inflammation score were determined blindly by two independent observers (30).
MEFs were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% Penicillin/streptomycin (Gibco). All cells tested negative for the presence of mycoplasma before experiments. The cells were treated with 10% CSE and 1 μg/ml LPS for the indicated time periods.
Lung tissue and cells were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (GenDEPOT). Total protein was quantified using the Bradford assay (Bio-Rad) according to the manufacturer's instructions and subjected to SDS-PAGE. Primary antibodies used were anti-HO-1 (sc-136960), anti-COX-2 (CST, #12282), anti-Tubulin (Sigma, T6074), anti-GAPDH (sc-25778), and anti-4-HNE (Abcam, ab46545). Specific bands were quantified by densitometry using NIH ImageJ.
Total RNA was isolated using the TRIzol reagent (Thermo-Fisher Scientific) according to the manufacturer's protocol. The complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the cDNA synthesis kit (Thermo-Fisher Scientific). Quantitative PCR was performed using SYBR Green (Applied Biosystems) and detected on the StepOnePlusTM Real-Time PCR system (Applied Biosystems). Primers used for the mouse genes are as follows.
All values are shown as mean ± standard error of mean (SEM). Differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA) and then determined with the least significant difference test. The criterion for significance was P < 0.05.
This work was supported by Basic Science Research Program (NRF-2018R1A2B6004112) to K.I.K. from the National Research Foundation of Korea (NRF), and by the Center for Women In Science, Engineering and Technology (WISET) Grant to J.J. from the Program for Returners into R&D, funded by the Ministry of Science and ICT (MSIT).
The authors have no conflicting interests.