Extracts of Actinobacillus actinomycetemcomitans induce apoptotic cell death in human osteoblastic MG63 cells.
Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts. The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 microg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 microg/mL protein. By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 microg/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.
Morimoto
Y
Y
Department of Oral Anatomy, Kyushu Dental College, Kitakyushu, Japan.
Morimoto
H
H
Murata
T
T
Kobayashi
S
S
Ohba
T
T
Haneji
T
T
D016475:3T3 Cells; D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000494:pharmacology*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003603:Cytotoxins / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000382:microbiology* / Q000502:physiology*; D018713:Prevotella intermedia / Q000737:chemistry; D013010:Sonication; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D003603:Cytotoxins; D008070:Lipopolysaccharides