Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells
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.
KeywordsAggregatibacter 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 (www.editage.jp) 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.
- 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.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
- 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.19126.96.36.1991 CrossRefPubMedGoogle Scholar
- 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
- 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.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
- 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.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
- 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
- 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
- 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
- 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.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
- 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
- 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.McClure R, Massari P (2014) TLR-dependent human mucosal epithelial cell responses to microbial pathogens. Front Immunol 12:386Google Scholar