Dust particles-induced intracellular Ca2+ signaling and reactive oxygen species in lung fibroblast cell line MRC5

Abstract

Epidemiologic interest in particulate matter (PM) is growing particularly because of its impact of respiratory health. It has been elucidated that PM evoked inflammatory signal in pulmonary epithelia. However, it has not been established Ca²⁺ signaling mechanisms involved in acute PM-derived signaling in pulmonary fibroblasts. In the present study, we explored dust particles PM modulated intracellular Ca²⁺ signaling and sought to provide a therapeutic strategy by antagonizing PM-induced intracellular Ca²⁺ signaling in human lung fibroblasts MRC5 cells. We demonstrated that PM10, less than 10 µm, induced intracellular Ca²⁺ signaling, which was mediated by extracellular Ca²⁺. The PM10-mediated intracellular Ca²⁺ signaling was attenuated by antioxidants, phospholipase blockers, polyADPR polymerase 1 inhibitor, and transient receptor potential melastatin 2 (TRPM2) inhibitors. In addition, PM-mediated increases in reactive oxygen species were attenuated by TRPM2 blockers, clotrimazole (CLZ) and N-(p-amylcinnamoyl) anthranilic acid (ACA). Our results showed that PM10 enhanced reactive oxygen species signal by measuring DCF fluorescence and the DCF signal attenuated by both TRPM2 blockers CLZ and ACA. Here, we suggest functional inhibition of TRPM2 channels as a potential therapeutic strategy for modulation of dust particle-mediated signaling and oxidative stress accompanying lung diseases.

Keywords: Calcium signaling; Lung fibroblast; Oxidative stress; Particulate matter; Reactive oxygen species.

Fig. 1. PM10-induced [Ca²⁺]_i signal by extracellular Ca²⁺ in human lung fibroblast MRC5 cells.

(A) Changes in [Ca²⁺]_i induced by 50 µg/mL PM10 in 1 mM Ca²⁺ medium (black line) and in Ca²⁺-free medium (gray line). Top bars indicate the extracellular solutions applied to MRC5 cells. (B) 50 µg/mL PM10-induced [Ca²⁺]_i signals were completely abolished by 10 µM BAPTA-AM. Traces shown were obtained from average signal except the stimulation with PM10 only. (C) ΔCa²⁺ was calculated at the indicated dotted line. Results are presented as mean±SEM. ^*p values of <0.01 were considered significant. (D) Cells were stimulated with the presence of La³⁺. Traces shown were obtained from average signal except the stimulation with PM10 only. (E) Results are presented as mean±SEM and ^*p values of <0.01 were considered significant. (F) Analysis of size distribution of dust particles.

Fig. 2. PM10-induced [Ca²⁺]_i signal is dependent on the PLC/IP₃ receptor pathway.

(A) Changes in [Ca²⁺]_i induced by 50 µg/mL PM10 in 1 mM Ca²⁺ medium. (B) 50 µg/mL PM10-induced [Ca²⁺]_i signals were prevented in MRC5 cells by 20 mM caffeine (an IP₃R antagonist). 50 µg/mL (C) PM10-induced [Ca²⁺]_i signaling in the presence of PLC inhibitor 5 µM U73122 or its inactive analog 5 µM U73343. The top bars indicate the types of extracellular solutions applied to cells. Traces shown were obtained from average signal except the co-stimulation with PM10 and U73343. (D) ΔCa²⁺ was calculated at the indicated dotted line. Results are presented as mean±SEM. ^*p values of <0.01 were considered significant.

Fig. 3. PM10-induced [Ca²⁺]_i signal is attenuated by inhibition of oxidative pathways.

(A) Changes in [Ca²⁺]_i induced by 50 µg/mL PM10. (B) 50 µg/mL PM10 induced [Ca²⁺]_i signaling in the presence of (A) 5 mM NAC (an anti-oxidant), (C) 10 µM 3-AB (a PARP-1 inhibitor), or (D) 10 µg/mL CLP. Top bars indicate the extracellular solution type applied. Traces shown were obtained from average signal. (E) ΔCa²⁺ was calculated at the indicated dotted line. Results are presented as mean±SEM. ^*p values of <0.01 were considered significant.

Fig. 4. PM10-induced [Ca²⁺]_i signal is required for the involvement of TRPM2 activation.

(A) Changes in [Ca²⁺]_i induced by 50 µg/mL PM10. 50 µg/mL PM10 induced [Ca²⁺]_i signaling in the presence of TRPM2 inhibitors: (B) 75 µM 2-APB, (C) 10 µM CLZ, or (D) 10 µM ACA. Top bars indicate the extracellular solution type applied to cells. Traces shown were obtained from average signal. (E) ΔCa²⁺ was calculated at the indicated dotted line. Results are presented as mean±SEM, and ^*p values of <0.01 were considered significant.

Fig. 5. PM10 induced ROS production and ROS levels were reduced in the presence of TRPM2 blockers.

The fluorescence of H2DCFDA was visualized by confocal laser scanning microscopy and quantified. (A) ROS production induced by 50 µg/mL PM10 and the effects of pretreating the TRPM2 blockers, 10 µM CLZ and 10 µM ACA. ROS images were taken within 60 min. (B) Results are presented as mean±SEM and ^*p values of <0.01 were considered significant. (C) ROS production in the presence of TRPM2 blockers CLZ and ACA. ^*p values of <0.01 were considered significant.