Molecular and Cellular Biochemistry

, Volume 436, Issue 1–2, pp 39–48 | Cite as

Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells

  • Shinsuke Kochi
  • Keisuke Yamashiro
  • Shoichi Hongo
  • Tadashi Yamamoto
  • Yuki Ugawa
  • Masayuki Shimoe
  • Mari Kawamura
  • Chiaki Hirata-Yoshihara
  • Hidetaka Ideguchi
  • Hiroshi Maeda
  • Shogo Takashiba


Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, β4, and β6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, β4, and β6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.


Aggregatibacter actinomycetemcomitans Cell adhesion Integrin Infection model Gingival epithelial cell Periodontal disease 



This study is supported by a Grant-in-Aid for Research Activity (Start-up Number: JP22890119) and a Grant-in-Aid for Young Scientists (B; Number: JP24792327). We would like to thank Editage ( for English language editing.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no duality of interest associated with this manuscript.

Supplementary material

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Supplementary material 1 (TIFF 46854 kb)
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Supplementary material 2 (TIFF 46854 kb)
11010_2017_3076_MOESM3_ESM.docx (28 kb)
Supplementary material 3 (DOCX 28 kb)


  1. 1.
    Kennedy JE, Polson AM (1973) Experimental marginal periodontitis in squirrel monkeys. J Periodontol 44:140–144. doi: 10.1902/jop.1973.44.3.140 CrossRefPubMedGoogle Scholar
  2. 2.
    Schroeder HE, Lindhe J (1980) Conditions and pathological features of rapidly destructive, experimental periodontitis in dogs. J Periodontol 51:6–19. doi: 10.1902/jop.1980.51.1.6 CrossRefPubMedGoogle Scholar
  3. 3.
    Bartold PM (2000) Cantley MD and Haynes DR (2010) Mechanisms and control of pathologic bone loss in periodontitis. Periodontology 53:55–69. doi: 10.1111/j.1600-0757.2010.00347.x CrossRefGoogle Scholar
  4. 4.
    Noguchi T, Shiba H, Komatsuzawa H, Mizuno N, Uchida Y, Ouhara K, Asakawa R, Kudo S, Kawaguchi H, Sugai M, Kurihara H (2003) Syntheses of prostaglandin E2 and E-cadherin and gene expression of beta-defensin-2 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans. Inflammation 27:341–349CrossRefPubMedGoogle Scholar
  5. 5.
    Uchida Y, Shiba H, Komatsuzawa H, Takemoto T, Sakata M, Fujita T, Kawaguchi H, Sugai M, Kurihara H (2001) Expression of IL-1 beta and IL-8 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans. Cytokine 14:152–161. doi: 10.1006/cyto.2001.0863 CrossRefPubMedGoogle Scholar
  6. 6.
    Delva E, Tucker DK, Kowalczyk AP (2009) The desmosome. Cold Spring Harb Perspect Biol 1:a002543. doi: 10.1101/cshperspect.a002543 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Myal Y, Leygue E, Blanchard AA (2010) Claudin 1 in breast tumorigenesis: revelation of a possible novel “claudin high” subset of breast cancers. J Biomed Biotechnol 2010:956897. doi: 10.1155/2010/956897 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nielsen MS, Axelsen LN, Sorgen PL, Verma V, Delmar M, Holstein-Rathlou NH (2012) Gap junctions. Compr Physiol 2:1981–2035. doi: 10.1002/cphy.c110051 PubMedGoogle Scholar
  9. 9.
    Satterfield MC, Dunlap KA, Hayashi K, Burghardt RC, Spencer TE, Bazer FW (2007) Tight and adherens junctions in the ovine uterus: differential regulation by pregnancy and progesterone. Endocrinology 148:3922–3931. doi: 10.1210/en.2007-0321 CrossRefPubMedGoogle Scholar
  10. 10.
    Katz J, Sambandam V, Wu JH, Michalek SM, Balkovetz DF (2000) Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes. Infect Immun 68:1441–1449CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kinumatsu T, Hashimoto S, Muramatsu T, Sasaki H, Jung HS, Yamada S, Shimono M (2009) Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells. J Periodontal Res 44:13–20. doi: 10.1111/j.1600-0765.2007.01036.x CrossRefPubMedGoogle Scholar
  12. 12.
    Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673–687CrossRefPubMedGoogle Scholar
  13. 13.
    Takada Y, Ye X, Simon S (2007) The integrins. Genome Biol 8:215. doi: 10.1186/gb-2007-8-5-215 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Gräber HG, Conrads G, Wilharm J, Lampert F (1999) Role of interactions between integrins and extracellular matrix components in healthy epithelial tissue and establishment of a long junctional epithelium during periodontal wound healing: a review. J Periodontol 70:1511–1522. doi: 10.1902/jop.1999.70.12.1511 CrossRefPubMedGoogle Scholar
  15. 15.
    Hongo S, Yamamoto T, Yamashiro K, Shimoe M, Tomikawa K, Ugawa Y, Kochi S, Ideguchi H, Maeda H, Takashiba S (2016) Smad2 overexpression enhances adhesion of gingival epithelial cells. Arch Oral Biol 71:46–53. doi: 10.1016/j.archoralbio.2016.06.025 CrossRefPubMedGoogle Scholar
  16. 16.
    Kwok T, Zabler D, Urman S, Rohde M, Hartig R, Wessler S, Misselwitz R, Berger J, Sewald N, König W, Backert S (2007) Helicobacter exploits integrin for type IV secretion and kinase activation. Nature 449:862–866. doi: 10.1038/nature06187 CrossRefPubMedGoogle Scholar
  17. 17.
    Nakagawa I, Amano A, Kuboniwa M, Nakamura T, Kawabata S, Hamada S (2002) Functional differences among FimA variants of Porphyromonas gingivalis and their effects on adhesion to and invasion of human epithelial cells. Infect Immun 70:277–285CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Fine DH, Kaplan JB (2000) Kachlany SC and Schreiner HC (2006) How we got attached to Actinobacillus actinomycetemcomitans: a model for infectious diseases. Periodontology 42:114–157. doi: 10.1111/j.1600-0757.2006.00189.x CrossRefGoogle Scholar
  19. 19.
    Asakawa R, Komatsuzawa H, Kawai T, Yamada S, Goncalves RB, Izumi S, Fujiwara T, Nakano Y, Suzuki N, Uchida Y, Ouhara K, Shiba H, Taubman MA, Kurihara H, Sugai M (2003) Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans. Mol Microbiol 50:1125–1139CrossRefPubMedGoogle Scholar
  20. 20.
    Kiley P, Holt SC (1980) Characterization of the lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4 and N27. Infect Immun 30:862–873PubMedPubMedCentralGoogle Scholar
  21. 21.
    Lally ET, Kieba IR, Demuth DR, Rosenbloom J, Golub EE, Taichman NS, Gibson CW (1989) Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene. Biochem Biophys Res Commun 159:256–262CrossRefPubMedGoogle Scholar
  22. 22.
    Sugai M, Kawamoto T, Pérès SY, Ueno Y, Komatsuzawa H, Fujiwara T, Kurihara H, Suginaka H, Oswald E (1998) The cell cycle-specific growth-inhibitory factor produced by Actinobacillus actinomycetemcomitans is a cytolethal distending toxin. Infect Immun 66:5008–5019PubMedPubMedCentralGoogle Scholar
  23. 23.
    Meyer DH, Sreenivasan PK, Fives-Taylor PM (1991) Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans. Infect Immun 59:2719–2726PubMedPubMedCentralGoogle Scholar
  24. 24.
    Xynogala I, Volgina A, DiRienzo JM, Korostoff J (2009) Evaluation of the humoral immune response to the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans Y4 in subjects with localized aggressive periodontitis. Oral Microbiol Immunol 24:116–123. doi: 10.1111/j.1399-302X.2008.00483.x CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Fujita T, Kishimoto A, Shiba H, Hayashida K, Kajiya M, Uchida Y, Matsuda S, Takeda K, Ouhara K, Kawaguchi H, Abiko Y, Kurihara H (2010) Irsogladine maleate regulates neutrophil migration and E-cadherin expression in gingival epithelium stimulated by Aggregatibacter actinomycetemcomitans. Biochem Pharmacol 79:1496–1505. doi: 10.1016/j.bcp.2010.01.017 CrossRefPubMedGoogle Scholar
  26. 26.
    Han YW, Shi W, Huang GT, Kinder Haake S, Park NH, Kuramitsu H, Genco RJ (2000) Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infect Immun 68:3140–3146CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Fujita T, Yumoto H, Shiba H, Ouhara K, Miyagawa T, Nagahara T, Matsuda S, Kawaguchi H, Matsuo T, Murakami S, Kurihara H (2012) Irsogladine maleate regulates epithelial barrier function in tumor necrosis factor-α-stimulated human gingival epithelial cells. J Periodontal Res 47:55–61. doi: 10.1111/j.1600-0765.2011.01404.x CrossRefPubMedGoogle Scholar
  28. 28.
    Wang SC, Yu M, Li YH, Piao HL, Tang CL, Sun C, Zhu R, Li MQ, Jin LP, Li DJ, Du MR (2013) Cyclosporin A promotes proliferating cell nuclear antigen expression and migration of human cytotrophoblast cells via the mitgen-activated protein kinase-3/1-mediated nuclear factor-κB signaling pathways. Int J Clin Exp Pathol 6:1999–2010PubMedPubMedCentralGoogle Scholar
  29. 29.
    Hirosue M, Kokeguchi S, Maeda H, Nishimura F, Takashiba S, Murayama Y (2001) Characterization of two genes encoding ferritin-like protein in Actinobacillus actinomycetemcomitans. Microbiol Immunol 45:721–727CrossRefPubMedGoogle Scholar
  30. 30.
    Ogura N, Akutsu M, Tobe M, Sakamaki H, Abiko Y, Kondoh T (2007) Microarray analysis of IL-1beta-stimulated chemokine genes in synovial fibroblasts from human TMJ. J Oral Pathol Med 36:223–228. doi: 10.1111/j.1600-0714.2007.00515.x CrossRefPubMedGoogle Scholar
  31. 31.
    Maeda T, Maeda H, Yamabe K, Mineshiba J, Tanimoto I, Yamamoto T, Naruishi K, Kokeguchi S, Takashiba S (2010) Highly expressed genes in a rough-colony-forming phenotype of Aggregatibacter actinomycetemcomitans: implication of a mip-like gene for the invasion of host tissue. FEMS Immunol Med Microbiol 58:226–236. doi: 10.1111/j.1574-695X.2009.00624.x CrossRefPubMedGoogle Scholar
  32. 32.
    Huang GT, Haake SK, Park NH (1998) Gingival epithelial cells increase interleukin-8 secretion in response to Actinobacillus actinomycetemcomitans challenge. J Periodontol 69:1105–1110. doi: 10.1902/jop.1998.69.10.1105 CrossRefPubMedGoogle Scholar
  33. 33.
    Winder AA, Wohlford-Lenane C, Scheetz TE, Nardy BN, Manzel LJ, Look DC, McCray PB (2009) Differential effects of cytokines and corticosteroids on toll-like receptor 2 expression and activity in human airway epithelia. Respir Res 10:96. doi: 10.1186/1465-9921-10-96 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Iwai H, Kim M, Yoshikawa Y, Ashida H, Ogawa M, Fujita Y, Muller D, Kirikae T, Jackson PK, Kotani S, Sasakawa C (2007) A bacterial effector targets Mad2L2, an APC inhibitor, to modulate host cell cycling. Cell 130:611–623. doi: 10.1016/j.cell.2007.06.043 CrossRefPubMedGoogle Scholar
  35. 35.
    Sugisawa M, Masaoka T, Enokiya Y, Muramatsu T, Hashimoto S, Yamada S, Shimono M (2010) Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium. J Periodontal Res 45:284–291. doi: 10.1111/j.1600-0765.2009.01231.x CrossRefPubMedGoogle Scholar
  36. 36.
    Larjava H, Koivisto L, Häkkinen L, Heino J (2011) Epithelial integrins with special reference to oral epithelia. J Dent Res 90:1367–1376. doi: 10.1177/0022034511402207 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Liu Z, Kobayashi K, van Dinther M, van Heiningen SH, Valdimarsdottir G, van Laar T, Scharpfenecker M, Löwik CW, Goumans MJ, Ten Dijke P, Pardali E (2009) VEGF and inhibitors of TGFbeta type-I receptor kinase synergistically promote blood-vessel formation by inducing alpha5-integrin expression. J Cell Sci 122:3294–3302. doi: 10.1242/jcs.048942 CrossRefPubMedGoogle Scholar
  38. 38.
    Xu JK, Chen HJ, Li XD, Huang ZL, Xu H, Yang HL, Hu J (2012) Optimal intensity shock wave promotes the adhesion and migration of rat osteoblasts via integrin β1-mediated expression of phosphorylated focal adhesion kinase. J Biol Chem 287:26200–26212. doi: 10.1074/jbc.M112.349811 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Urbich C, Dernbach E, Reissner A, Vasa M, Zeiher AM, Dimmeler S (2002) Shear stress-induced endothelial cell migration involves integrin signaling via the fibronectin receptor subunits alpha(5) and beta(1). Arterioscler Thromb Vasc Biol 22:69–75CrossRefPubMedGoogle Scholar
  40. 40.
    Maldonado BA, Furcht LT (1995) Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide. Invest Ophthalmol Vis Sci 36:2120–2126PubMedGoogle Scholar
  41. 41.
    Tsuda K, Furuta N, Inaba H, Kawai S, Hanada K, Yoshimori T, Amano A (2008) Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles. Cell Struct Funct 33:123–132CrossRefPubMedGoogle Scholar
  42. 42.
    Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S (1999) Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162:3749–3752PubMedGoogle Scholar
  43. 43.
    Madeira MF, Queiroz-Junior CM, Cisalpino D, Werneck SM, Kikuchi H, Fujise O, Ryffel B, Silva TA, Teixeira MM, Souza DG (2013) MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice. Mol Oral Microbiol 28:415–424. doi: 10.1111/omi.12034 CrossRefPubMedGoogle Scholar
  44. 44.
    McClure R, Massari P (2014) TLR-dependent human mucosal epithelial cell responses to microbial pathogens. Front Immunol 12:386Google Scholar
  45. 45.
    Haubek D, Ennibi OK, Poulsen K, Vaeth M, Poulsen S, Kilian M (2008) Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study. Lancet 19:237–242CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Shinsuke Kochi
    • 1
  • Keisuke Yamashiro
    • 1
  • Shoichi Hongo
    • 1
  • Tadashi Yamamoto
    • 2
  • Yuki Ugawa
    • 1
  • Masayuki Shimoe
    • 1
  • Mari Kawamura
    • 1
  • Chiaki Hirata-Yoshihara
    • 1
  • Hidetaka Ideguchi
    • 1
  • Hiroshi Maeda
    • 3
  • Shogo Takashiba
    • 1
  1. 1.Department of Pathophysiology-Periodontal ScienceOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesOkayamaJapan
  2. 2.Department of Periodontics and EndodonticsOkayama University HospitalOkayamaJapan
  3. 3.Deparment of EndodonticsOsaka Dental UniversityOsakaJapan

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