title: [Expression of Bim, Bax and Bak in the process of gingipain-induced osteoblast apoptosis].
abstract: OBJECTIVE
  To establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak).
  
  
  METHODS
  Gingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis.
  
  
  RESULTS
  Arginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1.
  
  
  CONCLUSIONS
  1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.
authors.affiliation: Department of Periodontology, Sun Yat-sen University, Guangzhou, China.
authors.forename: Yu-ting
authors.initials: YT
authors.lastname: Chen
authors.forename: Xiang-chen
authors.initials: XC
authors.lastname: Song
authors.forename: Fu-ping
authors.initials: FP
authors.lastname: Zhang
authors.forename: Min
authors.initials: M
authors.lastname: Liang
mesh_terms: D018829:Adhesins, Bacterial / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D000080867:Gingipain Cysteine Endopeptidases; D006801:Humans; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D018829:Adhesins, Bacterial; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D000080867:Gingipain Cysteine Endopeptidases; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D014107:Tosyllysine Chloromethyl Ketone; D003546:Cysteine Endopeptidases