Abstract
Objective
In this study, we investigated the molecular basis of reactive oxygen species (ROS) generation induced by lipopolysaccharide (LPS) in A549 cells—an alveolar epithelial cell line.
Experimental design
A549 cells or normal human bronchial epithelial (NHBE) cells were stimulated with LPS. ROS generation was measured in A549 cells or NHBE cells pre-treated with a selective inhibitor of phosphatidylinositol 3-kinase γ (PI3Kγ), AS 605240, PI3Kγ siRNA, or a ROS scavenger, pyridoxamine (PM).
Results
Treatment of A549 cells or NHBE cells with LPS caused a significant increase in intracellular ROS generation. Pretreatment with the PI3Kγ inhibitor, AS 605240 decreased the LPS-induced increase of ROS generation, phosphorylation of Akt, and production of phosphatidyl 3,4,5-trisphosphate in A549 cells. In addition, interference with siRNA for PI3Kγ significantly reduced LPS-induced ROS generation in A549 cells. Treatment of A549 cells with LPS or hydrogen peroxide increased the nuclear factor-κB (NF-κB) in the nucleus, accompanying an increase in phosphorylation of inhibitory κB-α, degradation of the protein, and reduction of cytosolic NF-κB. Pretreatment with AS 605240 reduced these LPS-induced changes. In addition, pretreatment with PM or N-acetyl cysteine resulted in inhibition of nuclear NF-κB activation.
Conclusion
These results suggest that PI3Kγ plays a key role in LPS-induced ROS generation in alveolar epithelial cells, thereby activating NF-κB.
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References
Christodoulopoulos P, Wright E, Frenkiel S, Luster A, Hamid Q. Monocyte chemotactic proteins in allergen-induced inflammation in the nasal mucosa: effect of topical corticosteroids. J Allergy Clin Immunol. 1999;103:1036–44.
Rahman I. Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J Biochem Mol Biol. 2003;36:95–109.
Zhang X, Shan P, Sasidhar M, Chupp GL, Flavell RA, Choi AM, et al. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediates hyperoxia-induced cell death in lung epithelium. Am J Respir Cell Mol Biol. 2003;28:305–15.
Haddad JJ, Land SC. Redox signaling-mediated regulation of lipopolysaccharide-induced proinflammatory cytokine biosynthesis in alveolar epithelial cells. Antioxid Redox Signal. 2002;4:179–93.
Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L, et al. Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science. 2000;287:1049–53.
Condliffe AM, Davidson K, Anderson KE, Ellson CD, Crabbe T, Okkenhaug K, et al. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils. Blood. 2005;106:1432–40.
Lee KS, Lee HK, Hayflick JS, Lee YC, Puri KD. Inhibition of phosphoinositide 3-kinase delta attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model. FASEB J. 2006;20:455–65.
Lee KS, Kim SR, Park SJ, Park HS, Min KH, Lee MH, et al. Hydrogen peroxide induces vascular permeability via regulation of vascular endothelial growth factor. Am J Respir Cell Mol Biol. 2006;35:190–7.
Rahman I, MacNee W. Role of oxidants/antioxidants in smoking-induced airways diseases. Free Radic Biol Med. 1999;21:669–81.
Kim H, Hwang JS, Woo CH, Kim EY, Kim TH, Cho KJ, et al. TNF-α-induced up-regulation of intercellular adhesion molecule-1 is regulated by a Rac-ROS-dependent cascade in human airway epithelial cells. Exp Mol Med. 2008;40:167–75.
Frey RS, Gao X, Javaid K, Siddiqui SS, Rahman A, Malik AB. Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells. J Biol Chem. 2006;281:16128–38.
Acknowledgments
We thank Prof. Mie-Jae Im (Chonbuk National University Medical School, Jeonju, South Korea) for critical reading of the manuscript. This work was supported by the Korea Healthcare Technology R&D Project, Ministry for Health and Welfare, Republic of Korea; Grant A084144 (to Yong Chul Lee) and Grant A111992 (to So Ri Kim).
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Responsible Editor: Liwu Li.
H. J. Kim and S. R. Kim contributed equally to this work.
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Kim, H.J., Kim, S.R., Park, J.K. et al. PI3Kγ activation is required for LPS-induced reactive oxygen species generation in respiratory epithelial cells. Inflamm. Res. 61, 1265–1272 (2012). https://doi.org/10.1007/s00011-012-0526-7
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DOI: https://doi.org/10.1007/s00011-012-0526-7