Molecular and Cellular Biochemistry

, Volume 452, Issue 1–2, pp 63–70 | Cite as

NFE2L2/NRF2, OGG1, and cytokine responses of human gingival keratinocytes against oxidative insults of various origin

  • Gökhan KasnakEmail author
  • Eija Könönen
  • Stina Syrjänen
  • Mervi Gürsoy
  • Fares Zeidán-Chuliá
  • Erhan Firatli
  • Ulvi K. Gürsoy



Bacterial or tobacco-related insults induce oxidative stress in gingival keratinocytes. The aim of this study was to investigate anti-oxidative and cytokine responses of human gingival keratinocytes (HMK cells) against Porphyromonas gingivalis lipopolysaccharide (Pg LPS), nicotine, and 4-nitroquinoline N-oxide (4-NQO).

Materials and methods

HMK cells were incubated with Pg LPS (1 µl/ml), nicotine (1.54 mM), and 4-NQO (1 µM) for 24 h. Intracellular and extracellular levels of interleukin (IL)-1β, IL-1 receptor antagonist (IL-1Ra), IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) were measured with the Luminex® xMAP™ technique, and nuclear factor, erythroid 2 like 2 (NFE2L2/NRF2) and 8-oxoguanine DNA glycosylase (OGG1) with Western blots. Data were statistically analyzed by two-way ANOVA with Bonferroni correction.


All tested oxidative stress inducers increased intracellular OGG1 levels, whereas only nicotine and 4-NQO induced NFE2L2/NRF2 levels. Nicotine, 4-NQO, and their combinational applications with Pg LPS induced the secretions of IL-1β and IL-1Ra, while that of IL-8 was inhibited by the presence of Pg LPS. MCP-1 secretion was suppressed by nicotine, alone and together with Pg LPS, while 4-NQO activated its secretion. Treatment of HMK cells with Pg LPS, nicotine, 4-NQO, or their combinations did not affect VEGF levels.


Pg LPS, nicotine, and 4-NQO induce oxidative stress and regulate anti-oxidative response and cytokine expressions in human gingival keratinocytes differently. These results may indicate that bacterial and tobacco-related insults regulate distinct pathways.


Reactive oxygen species Anti-oxidative response Porphyromonas gingivalis Lipopolysaccharide Nicotine 4-Nitroquinoline N-oxide 



The authors are grateful to Katja Sampalahti and Oona Hällfors from the Institute of Dentistry, University of Turku, Finland, for their excellent technical assistance in cytokine level analyses. This study was supported by the Turku University Foundation (Grant No: 12198, UKG), Finnish Dental Society Apollonia (UKG), and by the Scientific and Technological Research Council of Turkey (Grant No: BIDEB 2219-1059B191600656, GK). FZC is grateful to FINDOS Turku Grant for Completing a Doctoral Degree (University of Turku), University of Turku Joint Research Grant Fund, and Selma and Maja-Lisa Selander's Fund (Minerva Foundation, Helsinki) for supporting this work.


  1. 1.
    Trivedi S, Lal N (2017) Antioxidant enzymes in periodontitis. J Oral Biol Craniofacial Res 7:54–57. CrossRefGoogle Scholar
  2. 2.
    Kasnak G, Firatli E, Könönen E, Olgac V, Zeidán-Chuliá F, Gursoy UK (2018) Elevated levels of 8-OHdG and PARK7/DJ-1 in peri-implantitis mucosa. Clin Implant Dent Relat Res. Google Scholar
  3. 3.
    Kanzaki H, Wada S, Narimiya T et al (2017) Pathways that regulate ROS scavenging enzymes, and their role in defense against tissue destruction in periodontitis. Front Physiol 8:351. CrossRefGoogle Scholar
  4. 4.
    Gölz L, Memmert S, Rath-Deschner B et al (2014) LPS from P. gingivalis and hypoxia increases oxidative stress in periodontal ligament fibroblasts and contributes to periodontitis. Mediat Inflamm 2014:986264. CrossRefGoogle Scholar
  5. 5.
    Nociti FH, Casati MZ, Duarte PM (2015) Current perspective of the impact of smoking on the progression and treatment of periodontitis. Periodontol 2000 67:187–210. CrossRefGoogle Scholar
  6. 6.
    Arima Y, Nishigori C, Takeuchi T et al (2006) 4-Nitroquinoline 1-oxide forms 8-hydroxydeoxyguanosine in human fibroblasts through reactive oxygen species. Toxicol Sci 91:382–392. CrossRefGoogle Scholar
  7. 7.
    Groeger SE, Meyle J (2015) Epithelial barrier and oral bacterial infection. Periodontol 2000 69:46–67. CrossRefGoogle Scholar
  8. 8.
    Yucel-Lindberg T, Båge T (2013) Inflammatory mediators in the pathogenesis of periodontitis. Expert Rev Mol Med 15:e7. CrossRefGoogle Scholar
  9. 9.
    Chapple ILC, Brock GR, Milward MR et al (2007) Compromised GCF total antioxidant capacity in periodontitis: cause or effect? J Clin Periodontol 34:103–110. CrossRefGoogle Scholar
  10. 10.
    Kataoka K, Ekuni D, Tomofuji T et al (2016) Visualization of oxidative stress induced by experimental periodontitis in keap1-dependent oxidative stress detector-luciferase mice. Int J Mol Sci 17:11. CrossRefGoogle Scholar
  11. 11.
    Chiu AV, Saigh M, Al McCulloch CA, Glogauer M (2017) The role of NrF2 in the regulation of periodontal health and disease. J Dent Res 96:975–983. CrossRefGoogle Scholar
  12. 12.
    Madankumar A, Tamilarasi S, Premkumar T et al (2017) Geraniol attenuates 4NQO-induced tongue carcinogenesis through downregulating the activation of NF-κB in rats. Mol Cell Biochem 434:7–15. CrossRefGoogle Scholar
  13. 13.
    Takano H, Momota Y, Kani K et al (2015) γ-Tocotrienol prevents 5-FU-induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5-FU-induced activation of Nrf2. Int J Oncol 46:1453–1460. CrossRefGoogle Scholar
  14. 14.
    Sima C, Aboodi GM, Lakschevitz FS et al (2016) Nuclear factor erythroid 2-related factor 2 down-regulation in oral neutrophils is associated with periodontal oxidative damage and severe chronic periodontitis. Am J Pathol 186:1417–1426. CrossRefGoogle Scholar
  15. 15.
    Park SY, Park DJ, Kim YH et al (2011) Schisandra chinensis α-iso-cubebenol induces heme oxygenase-1 expression through PI3K/Akt and Nrf2 signaling and has anti-inflammatory activity in Porphyromonas gingivalis lipopolysaccharide-stimulated macrophages. Int Immunopharmacol 11:1907–1915. CrossRefGoogle Scholar
  16. 16.
    Lee H-J, Pi S-H, Kim Y et al (2009) Effects of nicotine on antioxidant defense enzymes and RANKL expression in human periodontal ligament cells. J Periodontol 80:1281–1288. CrossRefGoogle Scholar
  17. 17.
    Mäkelä M, Larjava H, Pirilä E et al (1999) Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes. Exp Cell Res 251:67–78. CrossRefGoogle Scholar
  18. 18.
    Nishioka K, Ohtsubo T, Oda H et al (1999) Expression and differential intracellular localization of two major forms of human 8-oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs. Mol Biol Cell 10:1637–1652. CrossRefGoogle Scholar
  19. 19.
    Lau A, Tian W, Whitman SA, Zhang DD (2013) The predicted molecular weight of Nrf2: it is what it is not. Antioxid Redox Signal 18:91–93. CrossRefGoogle Scholar
  20. 20.
    Cheng R, Choudhury D, Liu C et al (2015) Gingival fibroblasts resist apoptosis in response to oxidative stress in a model of periodontal diseases. Nat Publ Group 146:1–8. Google Scholar
  21. 21.
    Imamura K, Kokubu E, Kita D et al (2015) Cigarette smoke condensate modulates migration of human gingival epithelial cells and their interactions with Porphyromonas gingivalis. J Periodontal Res 50:411–421. CrossRefGoogle Scholar
  22. 22.
    Zhou X, Zhuang Z, Wang W et al (2016) OGG1 is essential in oxidative stress induced DNA demethylation. Cell Signal 28:1163–1171. CrossRefGoogle Scholar
  23. 23.
    Cao H, Wang L, Chen B et al (2016) DNA demethylation upregulated Nrf2 expression in Alzheimer’s disease cellular model. Front Aging Neurosci 7:244. CrossRefGoogle Scholar
  24. 24.
    Jo Y, Lee B, Joo M, Hong C (2016) Nrf2 expression is upregulated in tumor infiltrating T cells and induces T cell anergy. J Immunol 196 (1 Supplement)143. Accessed 12 Feb 2018
  25. 25.
    Wang Y, Zhou Y, Graves DT (2014) FOXO transcription factors: their clinical significance and regulation. Biomed Res Int 2014:925350. Google Scholar
  26. 26.
    Wang Q, Sztukowska M, Ojo A et al (2015) FOXO responses to Porphyromonas gingivalis in epithelial cells. Cell Microbiol 17:1605–1617. CrossRefGoogle Scholar
  27. 27.
    Johnson GK, Guthmiller JM (2007) The impact of cigarette smoking on periodontal disease and treatment. Periodontol 2000 44:178–194. CrossRefGoogle Scholar
  28. 28.
    Kusumoto Y, Hirano H, Saitoh K et al (2004) Human gingival epithelial cells produce chemotactic factors interleukin-8 and monocyte chemoattractant protein-1 after stimulation with Porphyromonas gingivalis via toll-like receptor 2. J Periodontol 75:370–379. CrossRefGoogle Scholar
  29. 29.
    Fitzsimmons TR, Ge S, Bartold PM (2017) Compromised inflammatory cytokine response to P. gingivalis LPS by fibroblasts from inflamed human gingiva. Clin Oral Investig. Google Scholar
  30. 30.
    Johnson GK, Guthmiller JM, Joly S et al (2010) Interleukin-1 and interleukin-8 in nicotine- and lipopolysaccharide-exposed gingival keratinocyte cultures. J Periodontal Res 45:583–588. Google Scholar
  31. 31.
    Kashiwagi Y, Yanagita M, Kojima Y et al (2012) Nicotine up-regulates IL-8 expression in human gingival epithelial cells following stimulation with IL-1β or P. gingivalis lipopolysaccharide via nicotinic acetylcholine receptor signalling. Arch Oral Biol 57:483–490. CrossRefGoogle Scholar
  32. 32.
    Kim MM, Glazer CA, Mambo E et al (2006) Head and neck cancer cell lines exhibit differential mitochondrial repair deficiency in response to 4NQO. Oral Oncol 42:201–207. CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Dentistry, University of TurkuTurkuFinland
  2. 2.Faculty of DentistryIstanbul UniversityIstanbulTurkey
  3. 3.Welfare DivisionOral HealthCity of TurkuFinland
  4. 4.Department of PathologyTurku University HospitalTurkuFinland
  5. 5.Departamento de Ciencias Biomédicas Básicas, Facultad de Ciencias Biomédicas y de la SaludUniversidad Europea de MadridMadridSpain

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