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 β (TGFβ) 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 TGFβ1-induced CCN2 protein expression in human gingival fibroblasts (HGFs). This study investigates whether phenytoin can induce CCN2 synthesis through activated latent TGFβ in HGFs and its mechanisms. METHODS: CCN2 synthesis, latent TGFβ1 activation, and cellular reactive oxygen species (ROS) generation in HGFs were studied using western blot analysis, a TGFβ1 Emax® ImmunoAssay System, and 2',7'-dichlorodihydrofluorescein diacetate (an oxidation-sensitive fluorescent probe), respectively. RESULTS: Phenytoin significantly stimulated CCN2 synthesis, latent TGFβ1 activation, and ROS generation in HGFs. Addition of an TGFβ-neutralizing antibody, TGFβ 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 TGFβ1 activation. Curcumin dose-dependently decreased phenytoin-induced TGFβ1 activation and CCN2 synthesis in HGFs. CONCLUSIONS: Our findings indicated that NOX4-derived ROS play pivotal roles in phenytoin-induced latent TGFβ1 activation. Molecular targeting the phenytoin/NOX4/ROS/TGFβ1 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 TGFβ1 activation.