Phospholipase C,p38/MAPK,and NF-κBmediated induction of MIP-3α/CCL20 by Porphyromonas gingivalis

Abstract

Macrophage inflammatory protein-3α/C-C chemokine ligand 20 (MIP-3α/CCL20) is an antimicrobial peptide that plays an important role in innate immunity. In addition to direct microbicidal effects, MIP-3α/CCL20 also exhibits cytokine-like functions that are critical during dendritic cell activation. The aim of the present study was to investigate further which signaling pathways are involved in the MIP-3α/CCL20 mRNA expression in response to whole-cell Porphyromonas gingivalis. Primary gingival epithelial cells (GECs) and the immortalized oral keratinocyte cell-line OKF6/TERT-2 were stimulated with whole-cell P. gingivalis. Prior to stimulation, GECs and OKF6/TERT-2 cells were pretreated with specific inhibitors for nuclear-factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and phosphatidylinositol-3-kinase (PI3K). In GECs and OKF6/TERT-2 cells, activation of NF-κB was examined after exposure to P. gingivalis. The gene expression of MIP-3α/CCL20 was significantly induced in response to P. gingivalis (P ≤ 0.05) compared to unstimulated control cells. This induction was specifically blocked when cells were pre-incubated with inhibitors for NF-κB, MAPK, and PLC (P ≤ 0.05), but not for PI3K. These results demonstrate that P. gingivalis induces the MIP-3α/CCL20 mRNA in a NF-κB-, PLC-, and MAPKdependent manner.

Keywords

C-C chemokine ligand 20 gingival epithelial cells mitogen-activated protein kinase nuclear factor kappa B phospholipase C

References

Dale BA Periodontal epithelium: a newly recognized role in health and disease. Periodontol 2000 2002; 30: 70-78.

Chung WO , Dommisch H. , Yin L. , Dale BA Expression of defensins in gingiva and their role in periodontal health and disease. Curr Pharm Des 2007; 13: 3073-3083.

Lehrer RI Primate defensins. Nat Rev Microbiol 2004; 2: 727-738.

Yang D. , Chen Q. , Hoover DM et al. Many chemokines including CCL20/MIP-3alpha display antimicrobial activity. J Leukoc Biol 2003; 74: 448-455.

Yang D. , Chertov O. , Bykovskaia SN et al. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999; 286: 525-528.

Yang D. , Chen Q. , Chertov O. , Oppenheim JJ Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol 2000; 68: 9-14.

Hoover DM , Boulegue C. , Yang D. et al. The structure of human macrophage inflammatory protein-3alpha /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding activities with human beta-defensins. J Biol Chem 2002; 277: 37647-37654.

Schutyser E. , Struyf S. , Van Damme J. The CC chemokine CCL20 and its receptor CCR6 . Cytokine Growth Factor Rev 2003; 14: 409-426.

Yang D. , Biragyn A. , Kwak LW , Oppenheim JJ Mammalian defensins in immunity: more than just microbicidal. Trends Immunol 2002; 23: 291-296.

10.

Socransky SS , Haffajee AD , Cugini MA , Smith C. , Kent Jr RL Microbial complexes in subgingival plaque . J Clin Periodontol 1998; 25: 134-144.

11.

Lamont RJ , Jenkinson HF Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis . Microbiol Mol Biol Rev 1998; 62: 1244-1263.

12.

Holt SC , Kesavalu L. , Walker S. , Genco CA Virulence factors of Porphyromonas gingivalis. Periodontol 2000 1999; 20: 168-238.

13.

Sandros J. , Papapanou PN , Nannmark U. , Dahlen G. Porphyromonas gingivalis invades human pocket epithelium in vitro. J Periodontal Res 1994; 29: 62-69.

14.

Rudney JD , Chen R. , Sedgewick GJ Intracellular Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in buccal epithelial cells collected from human subjects. Infect Immun 2001; 69: 2700-2707.

15.

Lamont RJ , Chan A. , Belton CM , Izutsu KT , Vasel D. , Weinberg A. Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun 1995; 63: 3878-3885.

16.

Hintermann E. , Haake SK , Christen U. , Sharabi A. , Quaranta V. Discrete proteolysis of focal contact and adherens junction components in Porphyromonas gingivalis-infected oral keratinocytes: a strategy for cell adhesion and migration disabling. Infect Immun 2002; 70: 5846-5856.

17.

Belton CM , Izutsu KT , Goodwin PC , Park Y. , Lamont RJ Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells. Cell Microbiol 1999; 1: 215-223.

18.

Dommisch H. , Chung WO , Rohani MG et al. Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis. Infect Immun 2007 ; 75: 4326-4333.

19.

Chung WO , Dale BA Innate immune response of oral and foreskin keratinocytes: utilization of different signaling pathways by various bacterial species. Infect Immun 2004; 72: 352-358.

20.

Shpacovitch VM , Brzoska T. , Buddenkotte J. et al. Agonists of proteinase-activated receptor 2 induce cytokine release and activation of nuclear transcription factor kappaB in human dermal microvascular endothelial cells. J Invest Dermatol 2002; 118: 380-385.

21.

Ye RD Regulation of nuclear factor kappaB activation by G-protein-coupled receptors . J Leukoc Biol 2001; 70: 839-848.

22.

Uehara A. , Sugawara Y. , Sasano T. , Takada H. , Sugawara S. Proinflammatory cytokines induce proteinase 3 as membrane-bound and secretory forms in human oral epithelial cells and antibodies to proteinase 3 activate the cells through protease-activated receptor-2. J Immunol 2004; 173: 4179-4189.

23.

Dickson MA , Hahn WC , Ino Y. et al. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 2000; 20: 1436-1447.

24.

Fujimura S. , Hirai K. , Shibata Y. , Nakayama K. , Nakamura T. Comparative properties of envelope-associated argininegingipains and lysine-gingipain of Porphyromonas gingivalis. FEMS Microbiol Lett 1998; 163: 173-179.

25.

Holzhausen M. , Spolidorio LC , Ellen RP et al. Protease-activated receptor-2 activation: a major role in the pathogenesis of Porphyromonas gingivalis infection. Am J Pathol 2006; 168: 1189-1199.

26.

Chung WO , Hansen SR , Rao D. , Dale BA Protease-activated receptor signaling increases epithelial antimicrobial peptide expression. J Immunol 2004; 173: 5165-5170.

27.

Pfaffl MW A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29: e45.

28.

Desloges N. , Schubert C. , Wolff MH , Rahaus M. Varicella-zoster virus infection induces the secretion of interleukin-8. Med Microbiol Immunol 2008; 197: 277-284.

29.

Wang Y. , Yang J. , Gao Y. , Dong LJ , Liu S. , Yao Z. Reciprocal regulation of 5-alpha-dihydrotestosterone, interleukin-6 and interleukin-8 during proliferation of epithelial ovarian carcinoma. Cancer Biol Ther 2007; 6: 864-871.

30.

Macfarlane SR , Sloss CM , Cameron P. , Kanke T. , McKenzie RC , Plevin R. The role of intracellular Ca2+ in the regulation of proteinase-activated receptor-2 mediated nuclear factor kappa B signalling in keratinocytes. Br J Pharmacol 2005; 145: 535-544.

31.

Yoshida N. , Katada K. , Handa O. et al. Interleukin-8 production via protease-activated receptor 2 in human esophageal epithelial cells. Int J Mol Med 2007; 19: 335-340.

32.

Goon Goh F. , Sloss CM , Cunningham MR , Nilsson M. , Cadalbert L. , Plevin R. G-protein-dependent and -independent pathways regulate proteinase-activated receptor-2 mediated p65 NFkappaB serine 536 phosphorylation in human keratinocytes. Cell Signal 2008; 20: 1267-1274.

33.

Vliagoftis H. , Schwingshackl A. , Milne CD et al. Proteinase-activated receptor-2-mediated matrix metalloproteinase-9 release from airway epithelial cells. J Allergy Clin Immunol 2000; 106: 537-545.

34.

Bretschneider E. , Kaufmann R. , Braun M. et al. Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells. Br J Pharmacol 1999; 126: 1735-1740.

35.

Foster N. , Cheetham J. , Taylor JJ , Preshaw PM VIP inhibits Porphyromonas gingivalis LPS-induced immune responses in human monocytes. J Dent Res 2005; 84: 999-1004.

36.

Brozovic S. , Sahoo R. , Barve S. et al. Porphyromonas gingivalis enhances FasL expression via up-regulation of NFkappaBmediated gene transcription and induces apoptotic cell death in human gingival epithelial cells. Microbiology 2006; 152: 797-806.

37.

Inomata M. , Into T. , Ishihara Y. , Nakashima M. , Noguchi T. , Matsushita K. Arginine-specific gingipain A from Porphyromonas gingivalis induces Weibel-Palade body exocytosis and enhanced activation of vascular endothelial cells through protease-activated receptors. Microbes Infect 2007; 9: 1500-1506.

38.

Yilmaz O. , Jungas T. , Verbeke P. , Ojcius DM Activation of the phosphatidylinositol 3-kinase/Akt pathway contributes to survival of primary epithelial cells infected with the periodontal pathogen Porphyromonas gingivalis. Infect Immun 2004 ; 72: 3743-3751.

39.

Duits LA , Ravensbergen B. , Rademaker M. , Hiemstra PS , Nibbering PH Expression of beta-defensin 1 and 2 mRNA by human monocytes, macrophages and dendritic cells. Immunology 2002; 106: 517-525.

40.

Biragyn A. , Ruffini PA , Leifer CA et al. Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science 2002; 298: 1025-1029.

41.

Marcet B. , Horckmans M. , Libert F. , Hassid S. , Boeynaems JM , Communi D. Extracellular nucleotides regulate CCL20 release from human primary airway epithelial cells, monocytes and monocyte-derived dendritic cells. J Cell Physiol 2007; 211: 716-727.

42.

Chung WO , Dale BA Differential utilization of nuclear factorkappaB signaling pathways for gingival epithelial cell responses to oral commensal and pathogenic bacteria. Oral Microbiol Immunol 2008; 23: 119-126.