Nicotine Induction of CCN2: from Smoking to Periodontal Fibrosis

Objectives:In clinical cases, gingival hyperplasia is often observed among smokers. However, there has been no report that indicates distinct relation between the thickening/fibrosis of periodontal tissue and habitual smoking.The aim of this study was to investigate the effects of nicotine on the production of a profibrotic molecule, connective tissue growth factor (CCN2/CTGF) in human gingival fibroblasts (HGFs) and periodontal ligament (PDL) cells.Methods:HGFs and PDL cells were isolated from normal periodontal tissues. Subconfluent cells were stimulated with 0 (control), 0.1, 1 or 10 µg/ml nicotine for 12-48 h. Effect of nicotine on CCN2/CTGF and type I collagen mRNA expression was evaluated by RT-PCR. Production/distribution of CCN2/CTGF protein in the cells was observed using immunofluorescence microscopy. Effect of nicotine on CCN2/CTGF, type I collagen, MMP-1 and TGF-ß1 production was quantitatively analyzed by ELISA. Cell growth assay and morphological analysis were also performed. Results: Proliferation of both cells and expression of Ccn2/Ctgf mRNA in HGFs were slightly increased at 0.1 µg/ml of nicotine (p<0.05). Interestingly, 1 µg/ml nicotine increased the production of CCN2/CTGF protein in both cells without increasing the mRNA expression (p<0.05). Immunofluorescence analysis revealed higher protein levels at 1 µg/ml in both cells compared to control. At 1 µg/ml, type I collagen mRNA (p<0.01), protein (p<0.05) and TGF-ß1 levels were increased, whereas MMP-1 was not significantly induced. Vacuolization and attenuated proliferation were observed at the same dose. Conclusion:This is the first study to show a relationship between CCN2/CTGF and nicotine in periodontium. Our previous study indicated that CCN2/CTGF is induced by TGF-ß1 (Takeuchi et al. 2008, in press). Since CCN2/CTGF is a profibrotic molecule, our results suggest that periodontal fibrosis is promoted by long-term synergistic induction of CCN2/CTGF by 2 distinct molecules in smokers; one is exogenous nicotine, and the other is endogenous TGF-ß1 from inflammatory tissues.