Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients

Abstract

Background/PurposeLysine-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.MethodsK6F 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.ResultsWe 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.ConclusionKgp 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.

Antiproliferative Activity and Induction of Apoptosis in Human Melanoma Cells by Houttuynia cordata Thunb Extract

“…The samples were centrifuged, and 100 μg of the protein was incubated with a reaction buffer and colorimetric tetrapeptides, Asp-Glu-ValAsp (DEVD)-p-nitroaniline (pNA) for caspase-3 and Ile-Glu-ThrAsp (IETD)-pNA for caspase-8, at 37˚C for 2 h. Optical density of MAPK measurement by ELISA. Cell lysates were prepared according to a previous method, with modifications (27). Briefly, cells were stimulated with HCT extracts for the indicated time periods, and were lysed by adding 120 μl of SDS sample buffer containing 50 mM dithiothreitol, 1 mM phenylmethane sulfonyl fluoride, and 0.5 mM Na 2 VO 3 ).…”

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts

“…containing 0.1% BSA with 10 ng/ml PDGF-BB. After a 24-hour incubation, the number of migrated cells was counted in 10 random fields under a microscope at ×40 magnification as described previously (63).…”

Noncanonical Activation of β-Catenin by Porphyromonas gingivalis

“…In addition, gingipains can cleave proMMP9, generating the mature active enzyme (52), which is important for cancer cell invasion and metastasis. Gingipains can also proteolytically process proteins on the epithelial cell surface, causing release and redistribution, with consequent effects on signal transduction and inflammatory re- sponses (53,54). Intracellularly, P. gingivalis gingipains can degrade mammalian target of rapamycin (mTOR), thus disrupting the mTOR pathway which regulates the cytoskeleton, as well as cleave ␤-actin directly (55,56).…”