Epigenetic regulation of TNFA expression in periodontal disease.
BACKGROUND
Tumor necrosis factor-α (TNF-α) plays a central role in the molecular pathogenesis of periodontal disease. However, the epigenetic regulation attributable to microbial and inflammatory signals at the biofilm-gingival interface are poorly understood. In this study, the DNA methylation alteration within the TNFA promoter in human gingival biopsies from different stages of periodontal disease is investigated and the regulatory mechanism of TNFA transcription by DNA methylation is explored.


METHODS
Gingival biopsies were obtained from 17 patients with chronic periodontitis (CP) and 18 periodontally healthy individuals. Another 11 individuals participated in an experimentally induced gingivitis study, and gingival biopsies were collected at the baseline, induction, and resolution phase. To confirm that TNFA promoter methylation modulated TNFA transcription, THP.1 cells were treated with a DNA methyltransferase inhibitor, 5-Aza-2-deoxycytidine (5-Aza-2dC), and an RAW294.7 cell line transfected with a TNFA promoter-specific luciferase reporter system with or without methylation was used.


RESULTS
In gingival biopsies from individuals with severe CP, two individual cytosine-guanine dinucleotides (CpG sites) within the TNFA promoter (at -163 and -161 bp) displayed increased methylation in CP samples compared to those with gingival health (16.1% ± 5.1% versus 11.0% ± 4.6%, P = 0.02 and 19.8% ± 4.1% versus 15.4% ± 3.6%, P = 0.04, respectively). The methylation level at -163 bp was inversely associated with the transcription level of TNFA (P = 0.018). However, no significant difference in the TNFA promoter methylation pattern was observed in samples biopsied during the induction or resolution phase of experimentally induced gingivitis, which represented a reversible periodontal lesion. THP.1 cells treated with 5-Aza-2dC demonstrated a time-dependent increase in TNFA messenger level. It was also found that the luciferase activity decreased 2.6-fold in a construct containing an in vitro methylated TNFA promoter when compared to the unmethylated insert (P = 0.03).


CONCLUSIONS
Although the biopsy samples represented a mixed cell population, the change in promoter methylation status in chronic periodontal disease suggested that DNA methylation may be an important regulatory mechanism in controlling TNFA transcriptional expression in periodontal disease.
Center for Oral and Systemic Diseases, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC.
Shaoping
S
Zhang
Silvana P
SP
Barros
Antonio J
AJ
Moretti
Ning
N
Yu
Jing
J
Zhou
John S
JS
Preisser
Mihai D
MD
Niculescu
Steven
S
Offenbacher
D000328:Adult; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001706:Biopsy; D044883:Campylobacter rectus / Q000276:immunology; D018929:Cell Culture Techniques; D002460:Cell Line; D055113:Chronic Periodontitis / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D018899:CpG Islands / Q000235:genetics; D003430:Cross-Sectional Studies; D019175:DNA Methylation / Q000235:genetics; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D005891:Gingivitis / Q000235:genetics / Q000276:immunology / Q000473:pathology; D006801:Humans; D008156:Luciferases; D049408:Luminescent Agents; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects / Q000276:immunology / Q000382:microbiology; D010010:Osteoclasts / Q000166:cytology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D055815:Young Adult
D049408:Luminescent Agents; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000077209:Decitabine; D008156:Luciferases; D015254:DNA Modification Methylases; D001374:Azacitidine