, Volume 37, Issue 2, pp 522–533 | Cite as

Beyond Toll-Like Receptors: Porphyromonas gingivalis Induces IL-6, IL-8, and VCAM-1 Expression Through NOD-Mediated NF-κB and ERK Signaling Pathways in Periodontal Fibroblasts

  • Jianru Liu
  • Yixiang Wang
  • Xiangying Ouyang


To investigate whether oligomerization domains (NODs) are involved in Porphyromonas gingivalis-induced interleukin (IL)-6, IL-8, and vascular cell adhesion molecule (VCAM)-1 expression beyond Toll-like receptors (TLRs), we investigated the role of NOD1/2 in P. gingivalis-induced IL-6, IL-8, and VCAM-1 expression in human gingival fibroblasts (hGFs) and periodontal ligament cells (hPDLCs). The mechanism was explored by activation and silence of NODs, electrophoretic mobility shift assay (EMSA), and pathway blockade assays. Results showed that P. gingivalis could induce NOD1, NOD2, IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLs at mRNA and protein levels. Activation of NOD1/2 by agonists could clearly upregulate the expression of these genes, while silence of NOD1/2 could remarkably attenuate them. EMSA and blockade of NF-κB and extracellular-signal-regulated kinase (ERK)1/2 pathway assays also verified that the two pathways were involved in NOD1/2-mediated IL-6, IL-8, and VCAM-1 expression. In conclusion, our findings demonstrated that P. gingivalis induced IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLCs through NOD1/2-mediated NF-κB and ERK1/2 signaling pathways beyond TLRs.


NOD1/2 VCAM-1 Periodontal fibroblasts NF-κB ERK1/2 



We thank Prof. Chenxiong Lai from the Kaohsiung Medical University in Taiwan for generously providing P. gingivalis W83. This study is supported by National Natural Science Foundation of China (no. 181271150).

Conflict of Interest

The authors declare there is no conflict of interests.

Supplementary material

10753_2013_9766_Fig6_ESM.jpg (237 kb)
Supplementary figure 1

Characterization of hPDLCs and hGFs. The fibroblasts were identified by immunohistochemistry of mesenchymal marker vimentin and epithelial markers cytokeratin (CK)-14. Both of hPDLCs and hGFs were typically spindle-shaped and were positive for the mesenchymal marker vimentin (C & D) and negative for the epithelial marker cytokeratin 14, CK (A & B). (JPEG 237 kb)

10753_2013_9766_MOESM1_ESM.tif (3.3 mb)
High resolution image (TIFF 3411 kb)
10753_2013_9766_Fig7_ESM.jpg (23 kb)
Supplementary figure 2

mRNA expression of TLR4 in P. gingivalis-challenged hPDLCs and hGFs. Cells were treated with P. gingivalis at indicated MOI for indicated time. Real-time PCR was applied to detect TLR4 expression. No significant change of its expression level was revealed before and after bacterial challenge (A & B). (JPEG 23 kb)

10753_2013_9766_MOESM2_ESM.tif (1.9 mb)
High resolution image (TIFF 1994 kb)


  1. 1.
    Socransky, S.S., A.D. Haffajee, M.A. Cugini, C. Smith, and R.L. Kent Jr. 1998. Microbial complexes in subgingival plaque. Journal of Clinical Periodontology 25(2): 134–144.PubMedCrossRefGoogle Scholar
  2. 2.
    Nishihara, T., and T. Koseki. 2004. Microbial etiology of periodontitis. Periodontology 2000(36): 14–26.CrossRefGoogle Scholar
  3. 3.
    Kim, Y.C., Y. Ko, S.D. Hong, K.Y. Kim, Y.H. Lee, C. Chae, and Y. Choi. 2010. Presence of Porphyromonas gingivalis and plasma cell dominance in gingival tissues with periodontitis. Oral Diseases 16(4): 375–381.PubMedCrossRefGoogle Scholar
  4. 4.
    Sun, Y., R. Shu, C.L. Li, and M.Z. Zhang. 2010. Gram-negative periodontal bacteria induce the activation of Toll-like receptors 2 and 4, and cytokine production in human periodontal ligament cells. Journal of Periodontology 81(10): 1488–1496.PubMedCrossRefGoogle Scholar
  5. 5.
    Chang, L.C., H.C. Kuo, S.F. Chang, H.J. Chen, K.F. Lee, T.H. Lin, T.Y. Huang, C.S. Choe, L.T. Lin, and C.N. Chen. 2013. Regulation of ICAM-1 expression in gingival fibroblasts infected with high-glucose-treated P. gingivalis. Cellular Microbiology 15(10): 1722–34. doi: 10.1111/cmi.12146.PubMedGoogle Scholar
  6. 6.
    Dongari-Bagtzoglou, A.I., and J.L. Ebersole. 1998. Increased presence of interleukin-6 (IL-6) and IL-8 secreting fibroblast subpopulations in adult periodontitis. Journal of Periodontology 69(8): 899–910.PubMedCrossRefGoogle Scholar
  7. 7.
    Osborn, L., C. Hession, R. Tizard, C. Vassallo, S. Luhowskyj, G. Chi-Rosso, and R. Lobb. 1989. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59(6): 1203–1211.PubMedCrossRefGoogle Scholar
  8. 8.
    Springer, T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76(2): 301–314.PubMedCrossRefGoogle Scholar
  9. 9.
    van Wetering, S., N. van den Berk, J.D. van Buul, F.P. Mul, I. Lommerse, R. Mous, J.P. ten Klooster, J.J. Zwaginga, and P.L. Hordijk. 2003. VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration. American Journal of Physiology. Cell physiology 285(2): C343–352.PubMedCrossRefGoogle Scholar
  10. 10.
    Klimiuk, P.A., M. Fiedorczyk, S. Sierakowski, and J. Chwiecko. 2007. Soluble cell adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin) in patients with early rheumatoid arthritis. Scandinavian Journal of Rheumatology 36(5): 345–350.PubMedCrossRefGoogle Scholar
  11. 11.
    Guray, U., A.R. Erbay, Y. Guray, M.B. Yilmaz, A.A. Boyaci, H. Sasmaz, S. Korkmaz, and E. Kutuk. 2004. Levels of soluble adhesion molecules in various clinical presentations of coronary atherosclerosis. International Journal of Cardiology 96(2): 235–240.PubMedCrossRefGoogle Scholar
  12. 12.
    Cybulsky, M.I., K. Iiyama, H. Li, S. Zhu, M. Chen, M. Iiyama, V. Davis, J.C. Gutierrez-Ramos, P.W. Connelly, and D.S. Milstone. 2001. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. The Journal of Clinical Investigation 107(10): 1255–1262.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Hannigan, E., D.P. O'Connell, A. Hannigan, and L.A. Buckley. 2004. Soluble cell adhesion molecules in gingival crevicular fluid in periodontal health and disease. Journal of Periodontology 75(4): 546–550.PubMedCrossRefGoogle Scholar
  14. 14.
    Lester, S.R., J.L. Bain, F.G. Serio, B.D. Harrelson, and R.B. Johnson. 2009. Relationship between gingival angiopoietin-1 concentrations and depth of the adjacent gingival sulcus. Journal of Periodontology 80(9): 1447–1453.PubMedCrossRefGoogle Scholar
  15. 15.
    Morandini, A.C., P.P. Chaves Souza, E.S. Ramos-Junior, D.T. Brozoski, C.R. Sipert, C.A. Souza Costa, and C.F. Santos. 2013. Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts. Journal of Periodontology 84(4): 535–544.PubMedCrossRefGoogle Scholar
  16. 16.
    Herath, T.D., R.P. Darveau, C.J. Seneviratne, C.Y. Wang, Y. Wang, and L. Jin. 2013. Tetra- and penta-acylated lipid A structures of Porphyromonas gingivalis LPS differentially activate TLR4-mediated NF-kappaB signal transduction cascade and immuno-inflammatory response in human gingival fibroblasts. PloS One 8(3): e58496.PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Hajishengallis, G., H. Sojar, R.J. Genco, and E. DeNardin. 2004. Intracellular signaling and cytokine induction upon interactions of Porphyromonas gingivalis fimbriae with pattern-recognition receptors. Immunological Investigations 33(2): 157–172.PubMedCrossRefGoogle Scholar
  18. 18.
    Inohara, Chamaillard, C. McDonald, and G. Nunez. 2005. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annual Review of Biochemistry 74: 355–383.PubMedCrossRefGoogle Scholar
  19. 19.
    Chung, W.O., J.Y. An, L. Yin, B.M. Hacker, M.G. Rohani, H. Dommisch, and D.H. DiJulio. 2010. Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria. Immunology Letters 131(2): 113–119.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Uehara, A., and H. Takada. 2007. Functional TLRs and NODs in human gingival fibroblasts. Journal of Dental Research 86(3): 249–254.PubMedCrossRefGoogle Scholar
  21. 21.
    Jeon, D.I., S.R. Park, M.Y. Ahn, S.G. Ahn, J.H. Park, and J.H. Yoon. 2012. NOD1 and NOD2 stimulation triggers innate immune responses of human periodontal ligament cells. International Journal of Molecular Medicine 29(4): 699–703.PubMedCentralPubMedGoogle Scholar
  22. 22.
    Nemoto, E., T. Honda, S. Kanaya, H. Takada, and H. Shimauchi. 2008. Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation. Journal of Periodontal Research 43(5): 585–593.PubMedCrossRefGoogle Scholar
  23. 23.
    Hosokawa, I., Y. Hosokawa, K. Ozaki, H. Yumoto, H. Nakae, and T. Matsuo. 2010. Proinflammatory effects of muramyldipeptide on human gingival fibroblasts. Journal of Periodontal Research 45(2): 193–199.PubMedCrossRefGoogle Scholar
  24. 24.
    Liu, J., J. Duan, Y. Wang, and X. Ouyang. 2013. ICAM-1 is Regulated by Porphyromonas Gingivalis Through NOD1 and NOD2 Molecules in Periodontal Fibroblasts. Journal of Periodontology. doi: 10.1902/jop.2013.130152.
  25. 25.
    Deshpande, R.G., M.B. Khan, and C.A. Genco. 1998. Invasion of aortic and heart endothelial cells by Porphyromonas gingivalis. Infection and Immunity 66(11): 5337–5343.PubMedCentralPubMedGoogle Scholar
  26. 26.
    \Zhou, S., J. Zhang, H. Zheng, Y. Zhou, F. Chen, and J. Lin. 2013. Inhibition of mechanical stress-induced NF-kappaB promotes bone formation. Oral Diseases 19(1): 59–64.PubMedCrossRefGoogle Scholar
  27. 27.
    Okugawa, T., T. Kaneko, A. Yoshimura, N. Silverman, and Y. Hara. 2010. NOD1 and NOD2 mediate sensing of periodontal pathogens. Journal of Dental Research 89(2): 186–191.PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Liu, R., T. Desta, M. Raptis, R.P. Darveau, and D.T. Graves. 2008. P. gingivalis and E. coli lipopolysaccharides exhibit different systemic but similar local induction of inflammatory markers. Journal of Periodontology 79(7): 1241–1247.Google Scholar
  29. 29.
    Katz, J., V. Sambandam, J.H. Wu, S.M. Michalek, and D.F. Balkovetz. 2000. Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes. Infection and Immunity 68(3): 1441–1449.PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Lin, S.J., Y.L. Chen, M.Y. Kuo, C.L. Li, and H.K. Lu. 2005. Measurement of gp130 cytokines oncostatin M and IL-6 in gingival crevicular fluid of patients with chronic periodontitis. Cytokine 30(4): 160–167.PubMedCrossRefGoogle Scholar
  31. 31.
    Garlet, G.P., M.J. Avila-Campos, C.M. Milanezi, B.R. Ferreira, and J.S. Silva. 2005. Actinobacillus actinomycetemcomitans-induced periodontal disease in mice: patterns of cytokine, chemokine, and chemokine receptor expression and leukocyte migration. Microbes and Infection/Institut Pasteur 7(4): 738–747.PubMedCrossRefGoogle Scholar
  32. 32.
    Bendre, M.S., D.C. Montague, T. Peery, N.S. Akel, D. Gaddy, and L.J. Suva. 2003. Interleukin-8 stimulation of osteoclastogenesis and bone resorption is a mechanism for the increased osteolysis of metastatic bone disease. Bone 33(1): 28–37.PubMedCrossRefGoogle Scholar
  33. 33.
    Joe, B.H., J.L. Borke, M. Keskintepe, P.J. Hanes, J.M. Mailhot, and B.B. Singh. 2001. Interleukin-1beta regulation of adhesion molecules on human gingival and periodontal ligament fibroblasts. Journal of Periodontology 72(7): 865–870.PubMedCrossRefGoogle Scholar
  34. 34.
    Hosokawa, Y., I. Hosokawa, K. Ozaki, H. Nakae, and T. Matsuo. 2006. Cytokines differentially regulate ICAM-1 and VCAM-1 expression on human gingival fibroblasts. Clinical and Experimental Immunology 144(3): 494–502.PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Hosokawa, Y., I. Hosokawa, K. Ozaki, H. Nakae, and T. Matsuo. 2006. Proinflammatory effects of tumour necrosis factor-like weak inducer of apoptosis (TWEAK) on human gingival fibroblasts. Clinical and Experimental Immunology 146(3): 540–549.PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Park, H.J., S.K. Jeong, S.R. Kim, S.K. Bae, W.S. Kim, S.D. Jin, T.H. Koo, H.O. Jang, I. Yun, K.W. Kim, and M.K. Bae. 2009. Resveratrol inhibits Porphyromonas gingivalis lipopolysaccharide-induced endothelial adhesion molecule expression by suppressing NF-kappaB activation. Archives of Pharmacal Research 32(4): 583–591.PubMedCrossRefGoogle Scholar
  37. 37.
    Chou, H.H., H. Yumoto, M. Davey, Y. Takahashi, T. Miyamoto, F.C. Gibson 3rd, and C.A. Genco. 2005. Porphyromonas gingivalis fimbria-dependent activation of inflammatory genes in human aortic endothelial cells. Infection and Immunity 73(9): 5367–5378.PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Murakami, S., Y. Shimabukuro, T. Saho, E. Hino, D. Kasai, T. Hashikawa, H. Hirano, and H. Okada. 1997. Immunoregulatory roles of adhesive interactions between lymphocytes and gingival fibroblasts. Journal of Periodontal Research 32(1 Pt 2): 110–114.PubMedCrossRefGoogle Scholar
  39. 39.
    Lu, X., E. Mu, Y. Wei, S. Riethdorf, Q. Yang, M. Yuan, J. Yan, Y. Hua, B.J. Tiede, B.G. Haffty, K. Pantel, J. Massague, and Y. Kang. 2011. VCAM-1 promotes osteolytic expansion of indolent bone micrometastasis of breast cancer by engaging alpha4beta1-positive osteoclast progenitors. Cancer Cell 20(6): 701–714.PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Tang, L., X.D. Zhou, Q. Wang, L. Zhang, Y. Wang, X.Y. Li, and D.M. Huang. 2011. Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts. Archives of Oral Biology 56(10): 1064–1072.PubMedCrossRefGoogle Scholar
  41. 41.
    Hitotsumatsu, O., R.C. Ahmad, R. Tavares, M. Wang, D. Philpott, E.E. Turer, B.L. Lee, N. Shiffin, R. Advincula, B.A. Malynn, C. Werts, and A. Ma. 2008. The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals. Immunity 28(3): 381–390.PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Kim, E.K., and E.J. Choi. 2010. Pathological roles of MAPK signaling pathways in human diseases. Biochimica et Biophysica Acta 1802(4): 396–405.PubMedCrossRefGoogle Scholar
  43. 43.
    Xu, Y., X. Xu, H. Jin, X. Yang, Q. Gu, and K. Liu. 2013. Effects of a thrombomodulin-derived peptide on monocyte adhesion and intercellular adhesion molecule-1 expression in lipopolysaccharide-induced endothelial cells. Molecular Vision 19: 203–212.PubMedCentralPubMedGoogle Scholar
  44. 44.
    Zhang, H., J. Chen, X. Liu, L. Awar, A. McMickle, F. Bai, S. Nagarajan, and S. Yu. 2013. IL-17 induces expression of vascular cell adhesion molecule through signalling pathway of NF-kappaB, but not Akt1 and TAK1 in vascular smooth muscle cells. Scandinavian Journal of Immunology 77(4): 230–237.PubMedCrossRefGoogle Scholar
  45. 45.
    Lee, S.J., S.C. Cho, E.J. Lee, S. Kim, S.B. Lee, J.H. Lim, Y.H. Choi, W.J. Kim, and S.K. Moon. 2013. Interleukin-20 promotes migration of bladder cancer cells through extracellular signal-regulated kinase (ERK)-mediated MMP-9 protein expression leading to nuclear factor (NF-kappaB) activation by inducing the up-regulation of p21(WAF1) protein expression. The Journal of Biological Chemistry 288(8): 5539–5552.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of PeriodontologyPeking University School and Hospital of StomatologyBeijingChina
  2. 2.Peking University School and Hospital of StomatologyBeijingChina

Personalised recommendations