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Extracts of Actinobacillus actinomycetemcomitans Induce Apoptotic Cell Death in Human Osteoblastic MG63 Cells

Department of Oral Anatomy, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan, Department of Dental Radiology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

H. Morimoto

Department of Oral Anatomy, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

T. Murata

Department of Oral Anatomy, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan, Department of Preventive Dentistry, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

S. Kobayashi

Department of Oral Anatomy, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

T. Ohba

Department of Dental Radiology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

T. Haneji

Department of Oral Anatomy, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

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 µg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 µg/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 µg/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.

Key Words: apoptosis • oral bacteria • osteoblast • A. actinomycetemcomitans

Journal of Dental Research, Vol. 78, No. 3, 735-742 (1999)
DOI: 10.1177/00220345990780030501


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