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