Lipopolysaccharide extracted from Porphyromonas gingivalis induces DNA hypermethylation of runt-related transcription factor 2 in human periodontal fibroblasts
Osamu Uehara, Yoshihiro Abiko, Masato Saitoh, Hiroshi Miyakawa, Futoshi Nakazawa*
Received: May 5, 2012 Revised: June 6, 2012 Accepted: August 6, 2012
Corresponding author. Division of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
Background and purpose:
Epigenetic alterations such as DNA methylation and histone acetylation are described as changes in the pattern of gene expression not involving the DNA sequence. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis has been shown to inhibit osteoblastic cell differentiation. We examined whether DNA hypermethylation was involved in the inhibitory effect of LPS on osteoblastic differentiation of fibroblasts derived from human periodontal ligament (HPDL).
The HPDL cells were incubated with LPS derived from P. gingivalis at a concentration of 10 μg/ml for 24 h. The cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza). Untreated cells were used as a control. Cell viability was determined using cell proliferation reagent. DNA methyltransferase (DNMT1) and runt-related transcription factor 2 (RUNX2) mRNAs were evaluated by quantitative polymerase chain reaction (RT-PCR). Analysis of RUNX2 DNA methylation was performed using quantitative methylation-specific PCR.
The expression level of RUNX2 was significantly lower in the cells stimulated with LPS than the controls. The presence of 5Aza increased the expression of RUNX2 in cells stimulated with LPS. The expression levels of DNMT1 mRNA in the cells stimulated with LPS were significantly higher than in the control. The presence of 5Aza completely abolished the upregulated expression of DNMT1 in cells stimulated with LPS. The methylation of DNA at 0.1 kb and −1.9 kb in the cells stimulated with LPS was significantly higher than the control.
The results indicate that DNA hypermethylation may be involved in the inhibitory effect of LPS on osteoblastic differentiation in HPDL.
Lipopolysaccharide, Methylation, Osteoblastic differentiation, Periodontal fibroblast, Runt-related transcription factor 2