IL-1β and TNF-α regulate mouse amelotin gene transcription in gingival epithelial cells

Amelotin (AMTN) is an enamel protein expressed in maturation-stage ameloblasts and junctional epithelium. To clarify the transcriptional regulation of the AMTN gene by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with luciferase constructs including various lengths of the mouse AMTN gene promoter, and gel shift and chromatin immunoprecipitation assays using mouse gingival epithelial GE1 cells. The levels of AMTN mRNA and protein in GE1 cells were increased after 6 h of stimulation with IL-1β (1 ng/mL) and TNF-α (10 ng/mL). IL-1β and TNF-α induced luciferase activities of the constructs between -116AMTN and -705AMTN including the mouse AMTN gene promoter. Transcriptional activation by IL-1β and TNF-α was partially inhibited in -460AMTN including 3-bp mutations in the CCAAT-enhancer-binding protein 1 (C/EBP1), C/EBP2 and Yin Yang 1 (YY1) elements. Transcriptional activities induced by IL-1β and TNF-α were inhibited by tyrosine kinase, MEK1/2 and PI3-kinase inhibitors. Results of ChIP assays showed that IL-1β and TNF-α increased C/EBPβ and YY1 binding to the C/EBP1, C/EBP2 and YY1 elements. These results demonstrate that IL-1β and TNF-α increase AMTN gene transcription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.