BKCa channel as a novel regulator of cellular DNA damage response in human bronchial epithelial cells in the presence of particulate matter

Abstract

While particulate matter (PM) is a well-studied genotoxic environmental agent, our understanding of the molecular mechanisms through which PM triggers its harmful health consequences remains insufficient. The respiratory epithelium serves as the primary site for the deposition of PM, thereby acting as a protective barrier. These epithelial cells are characterized by the presence of notable potassium channels, which are critical for the regulation of the fluid layer. In human bronchial epithelial cells (HBE), the large-conductance Ca²⁺-regulated potassium (BK_Ca) channels, localized to the apical site of the plasma membrane, are critical for the maintenance of proper airway surface liquid volume. In this work, we focused on the role of the BK_Ca channel and its potential role in DNA damage response (DDR) after PM exposure. The mechanisms of DDR have been extensively studied, however, the involvement of ion channels in this phenomenon is not known. Therefore, we used depleted for the BK_Ca channel HBE cells (HBE Δα BK_Ca) as a physiological model. We demonstrated that exposure to standardized PM in HBE Δα BK_Ca cells induced reduced clone formation capabilities, an increase in ROS levels, PARP1-dependent apoptosis, cell cycle changes, and an increase in DNA double-strand breaks. A gene expression assessment by qPCR analysis revealed changes in expression levels of genes encoding proteins, especially from the DNA-single strand breaks repair pathway involved in oxidative DNA damage repair. Our findings imply that the absence of the BK_Ca channel might weaken the cellular response to DNA damage, potentially making cells more susceptible to PM-induced genomic instability. In conclusion, our research indicates the novel role of the BK_Ca channel in DDR for the first time.

Keywords: Apoptosis; BKCa channel; DNA damage response; DNA-DSBs; DNA-SSBs; PARP.

Fig. 1

Clonogenic survival of HBE wt cells vs. HBE Δα BK_Ca after cytotoxic 24-hour treatment with PM (0, 30, 50, and 100 µg/ml). A: The representative images of colonies stained with Coomassie blue for nontreated and after 24-hour treatment of HBE cells with PM 50 µg/ml. B: The panel represents decreased colony survival fractions of HBE cells exposed to PM (SRM-2786) in comparison to the unexposed HBE cells. The colonies were analyzed after 9 days of incubation in 5% CO₂ and 37 °C and counted using countPHICS software. Survival Fraction (SF) was considered using the formula: SF = (plating efficiency of tested cells/plating efficiency of control cells x 100%). Data were expressed as a percentage, the point bars represent the mean ± SEM (n = 3). One-way ANOVA was used to analyze experimental data. P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns – not significant.

Fig. 2

The influence of PM exposure (50 µg/ml, 24 h) on HBE wt cells vs. HBE Δα BK_Ca on intracellular ROS level (in arbitrary units) and cleaved PARP apoptosis. A: Data represent total ROS measurements and are expressed as mean ± SD (n = 3) for ROS level analysis (H2DCFDA probe). B: Representative images of flow cytometry analysis PARP1 cleavage. The fragment of cleaved-PARP1 (89 kDa) was used as a marker of apoptosis (PE mouse anti-cleaved PARP (Asp214) antibody). C: The data were expressed as a percentage, the point bars represent the mean ± SEM (n = 3) for apoptosis. D: Western blot analysis of PARP1 protein expression levels (left panel). The anti-PARP1 antibody detects both the full-length PARP1 protein and its 89-kDa cleaved fragment. β-actin served as the loading control. E: Quantitative analysis (optical density) (right panel). The bar graphs show the levels of cleaved PARP1, based on pooled densitometric analyses (mean ± SEM, n = 3). Results are expressed as the ratio of cleaved protein to total protein, normalized accordingly. One-way ANOVA was used to analyze experimental data. P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns – not significant.

Fig. 3

Flow cytometry identification of cell cycle changes with anti-BrdU antibody in HBE wt and HBE Δα BK_Ca cells after PM exposure (50 µg/ml, 24 h). The cells were labeled with 10 µM BrdU for 1 h and stained with BrdU PerCP-Cy 5.5 antibody. Flow cytometry representative images (A panel) of DAPI versus BrdU PerCP-Cy 5.5 staining profile (% of HBE cells in G2/M phase) and relative quantification (B panel). Cells were properly gated: G0/G1 phase, S phase and G2/M phase. 50 µM etoposide (ETOP) was used as a positive control (2-hour incubation). Data were demonstrated as percentages of HBE cells and the bars correspond to the mean ± SEM (n = 3). One-way ANOVA was used to analyze experimental data. P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns – not significant.

Fig. 4

Identification of DNA damage in HBE cells after PM exposure (50 µg/ml, 24 h) with antibody to histone H2 AX (pS139). A: Flow cytometry representative images of BrdU PerCP-Cy 5.5 versus H2 AX (pS139) Alexa Fluor 647 profile (% indicating the proportion of DNA double-stranded breaks in presented experiment). BrdU-positive cells are color-gated red and DNA damage is dark blue. B: Relative quantification from three independent experiments. Levels of γH2 AX were normalized to untreated HBE wt control set to unity and the bars represent the mean ± SEM (n = 3). 50 µM etoposide (ETOP) was used as a positive control (2-hour incubation). One-way ANOVA was used to analyze experimental data. P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns – not significant.

Fig. 5

Cluster heatmap analysis of the normalized expression of DNA-damage signaling genes after PM exposure (50 µg/ml, 24 h). A: HBE wt vs. wt + PM; B: Δα BK_Ca vs. wt; C: Δα BK_Ca vs. Δα BK_Ca + PM; D: wt + PM vs. Δα BK_Ca + PM. The red color characterizes a relatively high level of gene expression whereas the green color indicates a low level. The data were analyzed (n = 3) and clustered by targets using the Reference Gene Selection Tool from CFX Maestro Software v2.3 (Bio-Rad Laboratories, Inc.).

Fig. 6

Relative to control the normalized expression of DNA-damage response and repair genes after PM exposure (50 µg/ml, 24 h). A: HBE wt vs. wt + PM; B: Δα BK_Ca vs. wt; C:. Δα BK_Ca vs. Δα BK_Ca + PM; D: wt + PM vs. Δα BK_Ca + PM. NER: the nucleotide excision repair, MMR: DNA mismatch repair, SSBR: single-strand breaks repair, DSBR: double-strand breaks repair. Bar plot of the relative expression of all genes calculated via the ΔΔCt method. The bars correspond to the mean ± SEM (n = 3). One-way ANOVA was used to analyze experimental data. P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns – not significant.