title: Cleavage of host cytokeratin-6 by lysine-specific gingipain induces gingival inflammation in periodontitis patients.
abstract: OBJECTIVE
  Lysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.
  
  
  METHODS
  K6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.
  
  
  RESULTS
  We identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359-378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.
  
  
  CONCLUSIONS
  Kgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.
authors: Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.
authors: Salunya
authors: S
authors: Tancharoen
authors: Department of Periodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors: Takashi
authors: T
authors: Matsuyama
authors: Laboratory of Functional Foods, Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, Japan.
authors: Ko-Ichi
authors: K
authors: Kawahara
authors: Membrane Protein and Ligand Analysis Center, Protosera Inc., Amagasaki, Japan.
authors: Kenji
authors: K
authors: Tanaka
authors: Membrane Protein and Ligand Analysis Center, Protosera Inc., Amagasaki, Japan.
authors: Lyang-Ja
authors: LJ
authors: Lee
authors: Department of Periodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors: Miho
authors: M
authors: Machigashira
authors: Department of Periodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors: Kazuyuki
authors: K
authors: Noguchi
authors: Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Science, Kagoshima, Japan.
authors: Takashi
authors: T
authors: Ito
authors: Department of Molecular Pathology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors: Takahisa
authors: T
authors: Imamura
authors: Department of Periodontics, Endodontics and Dental Hygiene, University of Louisville School of Dentistry, Louisville, Kentucky, United States of America; Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
authors: Jan
authors: J
authors: Potempa
authors: Department of Physiology, Kurume University School of Medicine, Fukuoka, Japan.
authors: Kiyoshi
authors: K
authors: Kikuchi
authors: Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Science, Kagoshima, Japan.
authors: Ikuro
authors: I
authors: Maruyama
mesh_terms: D018829:Adhesins, Bacterial / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D000080867:Gingipain Cysteine Endopeptidases; D005881:Gingiva / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005883:Gingival Crevicular Fluid / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D053553:Keratin-6 / Q000378:metabolism*; D010518:Periodontitis / Q000378:metabolism* / Q000473:pathology; D016966:Porphyromonas gingivalis; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D018829:Adhesins, Bacterial; D016207:Cytokines; D000080867:Gingipain Cysteine Endopeptidases; D053553:Keratin-6; D003546:Cysteine Endopeptidases