PURPOSE: Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-β1 (TGF-β1). METHODS: Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-β1. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification. RESULTS: We found that 5-aza enhanced TGF-β1-induced interleukin-11 (IL11) expression in gingival fibroblasts 2.37-fold (P=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 (PRG4) and NADPH oxidase 4 (NOX4). Consistent with this, 5-aza caused demethylation of the IL11 gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-β type I receptor kinase inhibitor SB431542 impeded the changes in IL11 expression, indicating that the effects of 5-aza require TGF-β signaling. 5-aza moderately increased the expression of TGF-β type II receptor (1.40-fold; P=0.009), possibly enhancing the responsiveness of fibroblasts to TGF-β1. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (P=0.005) and DNMT3B (P=0.002), which are enzymes responsible for gene methylation. CONCLUSIONS: These in vitro data suggest that the inhibition of DNA methylation by 5-aza supports TGF-β-induced IL11 expression in gingival fibroblasts.
DNA Methylation* ; DNA* ; Epigenomics ; Fibroblasts* ; Gene Expression ; Guanosine ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Interleukin-11* ; Methylation ; Methyltransferases ; NADPH Oxidase ; Phosphotransferases ; Polymerase Chain Reaction ; Proteoglycans ; Transforming Growth Factor beta1 ; Wound Healing

Periodontal disease is associated with chronic oxidative stress and inflammation.Caffeic acid phenethyl ester (CAPE),which is a potent inducer of heme oxygenase 1 (HO1),is a central active component of propolis,and the application of propolis improves periodontal status in diabetic patients.Here,primary murine macrophages were exposed to CAPE.Target gene expression was assessed by whole-genome microarray,RT-PCR and Western blotting.The antioxidative and anti-inflammatory activities of CAPE were examined by exposure of the cells to hydrogen peroxide,saliva and periodontal pathogens.The involvement of HO1 was investigated with the HO1 inhibitor tin protoporphyrin (SnPP) and knockout mice for Nrf2,which is a transcription factor for detoxifying enzymes.CAPE increased HO1 and other heat shock proteins in murine macrophages.A p38 MAPK inhibitor and Nrf2 knockout attenuated CAPE-induced HO1 expression in macrophages.CAPE exerted strong antioxidative activity.Additionally,CAPE reduced the inflammatory response to saliva and periodontal pathogens.Blocking HO1 decreased the antioxidative activity and attenuated the anti-inflammatory activity of CAPE.In conclusion,CAPE exerted its antioxidative effects through the Nrf2-mediated HO1 pathway and its anti-inflammatory effects through NF-κB inhibition.However,preclinical models evaluating the use of CAPE in periodontal inflammation are necessary in future studies.

Periodontal disease is associated with chronic oxidative stress and inflammation. Caffeic acid phenethyl ester (CAPE), which is a potent inducer of heme oxygenase 1 (HO1), is a central active component of propolis, and the application of propolis improves periodontal status in diabetic patients. Here, primary murine macrophages were exposed to CAPE. Target gene expression was assessed by whole-genome microarray, RT-PCR and Western blotting. The antioxidative and anti-inflammatory activities of CAPE were examined by exposure of the cells to hydrogen peroxide, saliva and periodontal pathogens. The involvement of HO1 was investigated with the HO1 inhibitor tin protoporphyrin (SnPP) and knockout mice for Nrf2, which is a transcription factor for detoxifying enzymes. CAPE increased HO1 and other heat shock proteins in murine macrophages. A p38 MAPK inhibitor and Nrf2 knockout attenuated CAPE-induced HO1 expression in macrophages. CAPE exerted strong antioxidative activity. Additionally, CAPE reduced the inflammatory response to saliva and periodontal pathogens. Blocking HO1 decreased the antioxidative activity and attenuated the anti-inflammatory activity of CAPE. In conclusion, CAPE exerted its antioxidative effects through the Nrf2-mediated HO1 pathway and its anti-inflammatory effects through NF-κB inhibition. However, preclinical models evaluating the use of CAPE in periodontal inflammation are necessary in future studies.
Animals ; Caffeic Acids ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Inflammation ; drug therapy ; Mice ; NF-kappa B ; antagonists & inhibitors ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Phenylethyl Alcohol ; analogs & derivatives ; pharmacology

Sclerostin is a Wnt signalling antagonist that controls bone metabolism. Sclerostin is expressed by osteocytes and cementocytes; however, its role in the formation of dental structures remains unclear. Here, we analysed the mandibles of sclerostin knockout mice to determine the influence of sclerostin on dental structures and dimensions using histomorphometry and micro-computed tomography (μCT) imaging. μCT and histomorphometric analyses were performed on the first lower molar and its surrounding structures in mice lacking a functional sclerostin gene and in wild-type controls. μCT on six animals in each group revealed that the dimension of the basal bone as well as the coronal and apical part of alveolar part increased in the sclerostin knockout mice. No significant differences were observed for the tooth and pulp chamber volume. Descriptive histomorphometric analyses of four wild-type and three sclerostin knockout mice demonstrated an increased width of the cementum and a concomitant moderate decrease in the periodontal space width. Taken together, these results suggest that the lack of sclerostin mainly alters the bone and cementum phenotypes rather than producing abnormalities in tooth structures such as dentin.
Animals ; Female ; Glycoproteins ; genetics ; Mice ; Mice, Knockout ; Periodontium ; metabolism ; Phenotype ; Tooth ; metabolism