Phenytoin induces connective tissue growth factor (CTGF/CCN2) production through NADPH oxidase 4-mediated latent TGFβ1 activation in human gingiva fibroblasts: Suppression by curcumin

Objective and Background Gingival overgrowth (GO) is a common side effect of some drugs such as anticonvulsants, immunosuppressant, and calcium channel blockers. Among them, the antiepileptic agent phenytoin is the most common agent related to this condition due to its high incidence. Transforming growth factor beta (TGF beta) importantly contributes to the pathogenesis of GO. Connective tissue growth factor (CTGF or CCN2) is a key mediator of tissue fibrosis and is positively associated with the degree of fibrosis in GO. We previously showed that Src, c-jun N-terminal kinase, and Smad3 mediate TGF beta 1-induced CCN2 protein expression in human gingival fibroblasts (HGFs). This study investigates whether phenytoin can induce CCN2 synthesis through activated latent TGF beta in HGFs and its mechanisms. Methods CCN2 synthesis, latent TGF beta 1 activation, and cellular reactive oxygen species (ROS) generation in HGFs were studied using western blot analysis, a TGF beta 1 Emax (R) ImmunoAssay System, and 2 ',7 '-dichlorodihydrofluorescein diacetate (an oxidation-sensitive fluorescent probe), respectively. Results Phenytoin significantly stimulated CCN2 synthesis, latent TGF beta 1 activation, and ROS generation in HGFs. Addition of an TGF beta-neutralizing antibody, TGF beta receptor kinase inhibitor SB431542, and Smad3 inhibitor SIS3 completely inhibited phenytoin-induced CCN2 synthesis. General antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor diphenylene iodonium, and specific NOX4 inhibitor plumbagin almost completely suppressed phenytoin-induced total cellular ROS and latent TGF beta 1 activation. Curcumin dose-dependently decreased phenytoin-induced TGF beta 1 activation and CCN2 synthesis in HGFs. Conclusions Our findings indicated that NOX4-derived ROS play pivotal roles in phenytoin-induced latent TGF beta 1 activation. Molecular targeting the phenytoin/NOX4/ROS/TGF beta 1 pathway may provide promising strategies for the prevention and treatment of GO. Curcumin-inhibited phenytoin-induced CCN2 synthesis is caused by the suppression of latent TGF beta 1 activation.