JNK/c-Jun signaling pathway mediates the fluoride-induced down-regulation of MMP-20 in vitro

Delayed removal of amelogenins, which are initially hydrolyzed by matrix metalloproteinase MMP-20, is a characteristic of enamel fluorosis. In this study, we investigated the regulation of MMP-20 and possible effects of fluoride on MMP-20 expression in human ameloblast lineage cells. Protein expression and signaling pathways of human ameloblast lineage cells, exposed to 10 μM fluoride, were compared to control cells without fluoride exposure. The role of activator protein-1 in MMP-20 regulation was analyzed by DNA-protein affinity precipitation and luciferase reporter gene assays. MMP-20 protein levels in human ameloblast lineage cells decreased in the presence of fluoride, while amelogenin and TIMP-2 were not altered. Fluoride also decreased the transcription of a luciferase reporter gene driven by the MMP-20 promoter. Down-regulation of MMP-20 by fluoride was related to suppression of JNK/c-Jun phosphorylation. In contrast, the JNK activator elevated the expression of MMP-20. Three c-Jun binding sites on the MMP-20 promoter were identified for the first time, and were occupied by c-Jun as MMP-20 was induced. Deletion of any one of AP-1 binding sites on the MMP-20 promoter significantly reduced the transcription of downstream luciferase reporter. These in vitro findings suggest that c-Jun is a key regulatory element for MMP-20 expression, and human ameloblast lineage cells can respond to fluoride by down-regulating MMP-20 transcription through the JNK/c-Jun signaling pathway.