Korean J Physiol Pharmacol.  2017 May;21(3):327-334. 10.4196/kjpp.2017.21.3.327.

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

• Lee DU
• Ji MJ
• Kang JY
• Kyung SY
• Hong JH

• KMID: 2376961
• DOI: http://doi.org/10.4196/kjpp.2017.21.3.327

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 Ca²âº signaling mechanisms involved in acute PM-derived signaling in pulmonary fibroblasts. In the present study, we explored dust particles PM modulated intracellular Ca²âº signaling and sought to provide a therapeutic strategy by antagonizing PM-induced intracellular Ca²âº signaling in human lung fibroblasts MRC5 cells. We demonstrated that PM10, less than 10 µm, induced intracellular Ca²âº signaling, which was mediated by extracellular Ca²âº. The PM10-mediated intracellular Ca²âº 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.

Keyword

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

MeSH Terms

Antioxidants
Calcium Signaling
Cell Line*
Clotrimazole
Dust*
Fibroblasts*
Fluorescence
Humans
Lung Diseases
Lung*
Oxidative Stress
Particulate Matter
Phospholipases
Reactive Oxygen Species*
Antioxidants
Clotrimazole
Dust
Particulate Matter
Phospholipases
Reactive Oxygen Species

Figure

• 

  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.

Reference

1. Kanatani KT, Ito I, Al-Delaimy WK, Adachi Y, Mathews WC, Ramsdell JW. Toyama Asian Desert Dust and Asthma Study Team. Desert dust exposure is associated with increased risk of asthma hospitalization in children. Am J Respir Crit Care Med. 2010; 182:1475–1481. PMID: 20656941.

2. He M, Ichinose T, Yoshida S, Yamamoto S, Inoue K, Takano H, Yanagisawa R, Nishikawa M, Mori I, Sun G, Shibamoto T. Asian sand dust enhances murine lung inflammation caused by Klebsiella pneumoniae. Toxicol Appl Pharmacol. 2012; 258:237–247. PMID: 22118940.

3. Yeo NK, Hwang YJ, Kim ST, Kwon HJ, Jang YJ. Asian sand dust enhances rhinovirus-induced cytokine secretion and viral replication in human nasal epithelial cells. Inhal Toxicol. 2010; 22:1038–1045. PMID: 20879958.

4. Wilker EH, Preis SR, Beiser AS, Wolf PA, Au R, Kloog I, Li W, Schwartz J, Koutrakis P, DeCarli C, Seshadri S, Mittleman MA. Long-term exposure to fine particulate matter, residential proximity to major roads and measures of brain structure. Stroke. 2015; 46:1161–1166. PMID: 25908455.

5. Shah AS, Lee KK, McAllister DA, Hunter A, Nair H, Whiteley W, Langrish JP, Newby DE, Mills NL. Short term exposure to air pollution and stroke: systematic review and meta-analysis. BMJ. 2015; 350:h1295. PMID: 25810496.

6. Blank F, Stumbles PA, Seydoux E, Holt PG, Fink A, Rothen-Rutishauser B, Strickland DH, von Garnier C. Size-dependent uptake of particles by pulmonary antigen-presenting cell populations and trafficking to regional lymph nodes. Am J Respir Cell Mol Biol. 2013; 49:67–77. PMID: 23492193.

7. Tsuda A, Rogers RA, Hydon PE, Butler JP. Chaotic mixing deep in the lung. Proc Natl Acad Sci U S A. 2002; 99:10173–10178. PMID: 12119385.

8. Oberdörster G. Pulmonary effects of inhaled ultrafine particles. Int Arch Occup Environ Health. 2001; 74:1–8. PMID: 11196075.

9. Philipson K, Falk R, Svartengren M, Jarvis N, Bailey M, Bergmann R, Hofmann W, Camner P. Does lung retention of inhaled particles depend on their geometric diameter? Exp Lung Res. 2000; 26:437–455. PMID: 11033767.

10. Choi HS, Ashitate Y, Lee JH, Kim SH, Matsui A, Insin N, Bawendi MG, Semmler-Behnke M, Frangioni JV, Tsuda A. Rapid translocation of nanoparticles from the lung airspaces to the body. Nat Biotechnol. 2010; 28:1300–1303. PMID: 21057497.

11. Mohammad AK, Amayreh LK, Mazzara JM, Reineke JJ. Rapid lymph accumulation of polystyrene nanoparticles following pulmonary administration. Pharm Res. 2013; 30:424–434. PMID: 22992832.

12. Ichinose T, Yoshida S, Hiyoshi K, Sadakane K, Takano H, Nishikawa M, Mori I, Yanagisawa R, Kawazato H, Yasuda A, Shibamoto T. The effects of microbial materials adhered to Asian sand dust on allergic lung inflammation. Arch Environ Contam Toxicol. 2008; 55:348–357. PMID: 18227959.

13. Kyung SY, Yoon JY, Kim YJ, Lee SP, Park JW, Jeong SH. Asian dust particles induce TGF-β(1) via reactive oxygen species in bronchial epithelial cells. Tuberc Respir Dis (Seoul). 2012; 73:84–92. PMID: 23166540.

14. Honda A, Matsuda Y, Murayama R, Tsuji K, Nishikawa M, Koike E, Yoshida S, Ichinose T, Takano H. Effects of Asian sand dust particles on the respiratory and immune system. J Appl Toxicol. 2014; 34:250–257. PMID: 23576315.

15. Kim ST, Ye MK, Shin SH. Effects of Asian sand dust on mucin gene expression and activation of nasal polyp epithelial cells. Am J Rhinol Allergy. 2011; 25:303–306. PMID: 22186242.

16. Shin SH, Ye MK, Hwang YJ, Kim ST. The effect of Asian sand dust-activated respiratory epithelial cells on activation and migration of eosinophils. Inhal Toxicol. 2013; 25:633–639. PMID: 24044679.

17. Cohen MD, Vaughan JM, Garrett B, Prophete C, Horton L, Sisco M, Kodavanti UP, Ward WO, Peltier RE, Zelikoff J, Chen LC. Acute high-level exposure to WTC particles alters expression of genes associated with oxidative stress and immune function in the lung. J Immunotoxicol. 2015; 12:140–153. PMID: 24911330.

18. Higashisaka K, Fujimura M, Taira M, Yoshida T, Tsunoda S, Baba T, Yamaguchi N, Nabeshi H, Yoshikawa T, Nasu M, Yoshioka Y, Tsutsumi Y. Asian dust particles induce macrophage inflammatory responses via mitogen-activated protein kinase activation and reactive oxygen species production. J Immunol Res. 2014; 2014:856154. DOI: 10.1155/2014/856154. PMID: 24987712.

19. Sakamoto N, Hayashi S, Gosselink J, Ishii H, Ishimatsu Y, Mukae H, Hogg JC, van Eeden SF. Calcium dependent and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells. Toxicol Appl Pharmacol. 2007; 225:134–141. PMID: 17720209.

20. Lansley AB, Sanderson MJ, Dirksen ER. Control of the beat cycle of respiratory tract cilia by Ca²⁺ and cAMP. Am J Physiol. 1992; 263:L232–L242. PMID: 1325130.

21. Hayashi T, Kawakami M, Sasaki S, Katsumata T, Mori H, Yoshida H, Nakahari T. ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium. Exp Physiol. 2005; 90:535–544. PMID: 15769883.

22. Abdullah LH, Conway JD, Cohn JA, Davis CW. Protein kinase C and Ca²⁺ activation of mucin secretion in airway goblet cells. Am J Physiol. 1997; 273:L201–L210. PMID: 9252557.

23. Davis CW. Regulation of mucin secretion from in vitro cellular models. Novartis Found Symp. 2002; 248:113–125. discussion 125-131, 277-282. PMID: 12568491.

24. Sakamoto Y, Ishijima M, Kaneko H, Kurebayashi N, Ichikawa N, Futami I, Kurosawa H, Arikawa-Hirasawa E. Distinct mechanosensitive Ca²⁺ influx mechanisms in human primary synovial fibroblasts. J Orthop Res. 2010; 28:859–864. PMID: 20108315.

25. Murata N, Ito S, Furuya K, Takahara N, Naruse K, Aso H, Kondo M, Sokabe M, Hasegawa Y. Ca²⁺ influx and ATP release mediated by mechanical stretch in human lung fibroblasts. Biochem Biophys Res Commun. 2014; 453:101–105. PMID: 25256743.

26. Wakui M, Osipchuk YV, Petersen OH. Receptor-activated cytoplasmic Ca²⁺ spiking mediated by inositol trisphosphate is due to Ca²⁺-induced Ca²⁺ release. Cell. 1990; 63:1025–1032. PMID: 1701691.

27. Ehrlich BE, Kaftan E, Bezprozvannaya S, Bezprozvanny I. The pharmacology of intracellular Ca²⁺-release channels. Trends Pharmacol Sci. 1994; 15:145–149. PMID: 7754532.

28. Criddle DN, Sutton R, Petersen OH. Role of Ca²⁺ in pancreatic cell death induced by alcohol metabolites. J Gastroenterol Hepatol. 2006; 21(Suppl 3):S14–S17. PMID: 16958662.

29. Wiegman CH, Michaeloudes C, Haji G, Narang P, Clarke CJ, Russell KE, Bao W, Pavlidis S, Barnes PJ, Kanerva J, Bittner A, Rao N, Murphy MP, Kirkham PA, Chung KF, Adcock IM. COPDMAP. Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease. J Allergy Clin Immunol. 2015; 136:769–780. PMID: 25828268.

30. Park L, Wang G, Moore J, Girouard H, Zhou P, Anrather J, Iadecola C. The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction. Nat Commun. 2014; 5:5318. PMID: 25351853.

31. Shekh K, Khan S, Jena G, Kansara BR, Kushwaha S. 3-Aminobenzamide--a PARP inhibitor enhances the sensitivity of peripheral blood micronucleus and comet assays in mice. Toxicol Mech Methods. 2014; 24:332–341. PMID: 24568202.

32. Choi SY, Kim YH, Lee YK, Kim KT. Chlorpromazine inhibits store-operated calcium entry and subsequent noradrenaline secretion in PC12 cells. Br J Pharmacol. 2001; 132:411–418. PMID: 11159689.

33. Mori Y, Kajimoto T, Nakao A, Takahashi N, Kiyonaka S. Receptor signaling integration by TRP channelsomes. Adv Exp Med Biol. 2011; 704:373–389. PMID: 21290307.

34. Takahashi N, Kozai D, Kobayashi R, Ebert M, Mori Y. Roles of TRPM2 in oxidative stress. Cell Calcium. 2011; 50:279–287. PMID: 21616534.

35. Nazıroğlu M, Özgül C, Çelik Ö, Çiğ B, Sözbir E. Aminoethoxydiphenyl borate and flufenamic acid inhibit Ca²⁺ influx through TRPM2 channels in rat dorsal root ganglion neurons activated by ADP-ribose and rotenone. J Membr Biol. 2011; 241:69–75. PMID: 21509529.

36. Roberge S, Roussel J, Andersson DC, Meli AC, Vidal B, Blandel F, Lanner JT, Le Guennec JY, Katz A, Westerblad H, Lacampagne A, Fauconnier J. TNF-α-mediated caspase-8 activation induces ROS production and TRPM2 activation in adult ventricular myocytes. Cardiovasc Res. 2014; 103:90–99. PMID: 24802330.

37. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003; 4:517–529. PMID: 12838335.

38. Lei B, Hitomi H, Mori T, Nagai Y, Deguchi K, Mori H, Masaki T, Nakano D, Kobori H, Kitaura Y, Nishiyama A. Effect of efonidipine on TGF-β1-induced cardiac fibrosis through Smad2-dependent pathway in rat cardiac fibroblasts. J Pharmacol Sci. 2011; 117:98–105. PMID: 21897055.

39. Ohyama T, Sato K, Kishimoto K, Yamazaki Y, Horiguchi N, Ichikawa T, Kakizaki S, Takagi H, Izumi T, Mori M. Azelnidipine is a calcium blocker that attenuates liver fibrosis and may increase antioxidant defence. Br J Pharmacol. 2012; 165:1173–1187. PMID: 21790536.

40. Mishima K, Maeshima A, Miya M, Sakurai N, Ikeuchi H, Hiromura K, Nojima Y. Involvement of N-type Ca²⁺ channels in the fibrotic process of the kidney in rats. Am J Physiol Renal Physiol. 2013; 304:F665–F673. PMID: 23324177.

41. Xu R, Li Q, Zhou XD, Perelman JM, Kolosov VP. Oxidative stress mediates the disruption of airway epithelial tight junctions through a TRPM2-PLCγ1-PKCα signaling pathway. Int J Mol Sci. 2013; 14:9475–9486. PMID: 23629676.

42. Horng T. Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome. Trends Immunol. 2014; 35:253–261. PMID: 24646829.

43. Li R, Kou X, Geng H, Xie J, Yang Z, Zhang Y, Cai Z, Dong C. Effect of ambient PM(2.5) on lung mitochondrial damage and fusion/fission gene expression in rats. Chem Res Toxicol. 2015; 28:408–418. PMID: 25560372.

44. Dellinger B, Pryor WA, Cueto R, Squadrito GL, Hegde V, Deutsch WA. Role of free radicals in the toxicity of airborne fine particulate matter. Chem Res Toxicol. 2001; 14:1371–1377. PMID: 11599928.

45. Kinnula VL, Fattman CL, Tan RJ, Oury TD. Oxidative stress in pulmonary fibrosis: a possible role for redox modulatory therapy. Am J Respir Crit Care Med. 2005; 172:417–422. PMID: 15894605.

46. Park JW, Yoon JY, Kim YJ, Kyung SY, Lee SP, Jeong SH, Moon C. Extracellular signal-regulated kinase (ERK) inhibition attenuates cigarette smoke extract (CSE) induced-death inducing signaling complex (DISC) formation in human lung fibroblasts (MRC-5) cells. J Toxicol Sci. 2010; 35:33–39. PMID: 20118622.

47. Park SA, Kim MJ, Park SY, Kim JS, Lee SJ, Woo HA, Kim DK, Nam JS, Sheen YY. EW-7197 inhibits hepatic, renal, and pulmonary fibrosis by blocking TGF-β/Smad and ROS signaling. Cell Mol Life Sci. 2015; 72:2023–2039. PMID: 25487606.

48. Janssen LJ, Mukherjee S, Ask K. Calcium homeostasis and ionic mechanisms in pulmonary fibroblasts. Am J Respir Cell Mol Biol. 2015; 53:135–148. PMID: 25785898.