Protease-active Extracellular Protein Preparations From Porphyromonas Gingivalis W83 Induce N-cadherin Proteolysis, Loss of Cell Adhesion, and Apoptosis in Human Epithelial Cells

J Periodontol. 2001 May;72(5):641-50. doi: 10.1902/jop.2001.72.5.641.

Abstract

Background: The protease-induced cytotoxicity of P. gingivalis may partly result from alteration of the extracellular matrix and/or surface receptors that mediate interaction between the host cells and their matrix. While P. gingivalis-induced degradation of E-cadherin has been documented, there is no information on the effects of P. gingivalis proteases on other members of this family of cell adhesion proteins.

Methods: Human epithelial KB cells were exposed to protease-active extracellular protein preparations from isogenic mutants of P. gingivalis. Quantification of apoptosis was performed by visualization of nuclei stained with 4,6'-diamidino-2-phenylindole. Alteration of cell adhesion proteins was examined by immunoblotting of cell lysates using monoclonal antibodies to those proteins.

Results: Treated cells exhibited loss of cell adhesion properties with apoptotic cell death subsequently observed. These effects correlated with the different levels of cysteine-dependent proteolytic activities of the isogenic mutants tested. Cleavage of N-cadherin was observed in immunoblots of lysates from detached cells. There was a direct correlation between the kinetics of N-cadherin cleavage and loss of cell adhesion properties. Loss of cell adhesion, as well as N-cadherin cleavage, could be inhibited by preincubation of P. gingivalis protease active extracellular protein preparations with the cysteine protease inhibitor TLCK. In control experiments, the cleavage of N-cadherin was detected after treatment of KB cells with trypsin but not after cell dissociation by a non-enzymatic method.

Conclusions: These results suggest that extracellular proteases from P. gingivalis can induce degradation of N-cadherin, which could have implications for the pathogenicity of this bacterium.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Analysis of Variance
  • Antibodies, Monoclonal
  • Apoptosis / drug effects*
  • Cadherins / metabolism*
  • Cell Adhesion / drug effects
  • Cell Nucleus / drug effects
  • Cysteine Endopeptidases / pharmacology
  • Cysteine Proteinase Inhibitors / pharmacology
  • Endopeptidases / pharmacology*
  • Epithelial Cells / drug effects
  • Fluorescent Dyes
  • Humans
  • Immunoblotting
  • Indoles
  • KB Cells / drug effects
  • Mutation / genetics
  • Porphyromonas gingivalis / enzymology*
  • Porphyromonas gingivalis / genetics
  • Porphyromonas gingivalis / pathogenicity
  • Tosyllysine Chloromethyl Ketone / pharmacology
  • Trypsin / pharmacology

Substances

  • Antibodies, Monoclonal
  • Cadherins
  • Cysteine Proteinase Inhibitors
  • Fluorescent Dyes
  • Indoles
  • Tosyllysine Chloromethyl Ketone
  • DAPI
  • Endopeptidases
  • Trypsin
  • Cysteine Endopeptidases