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

Abstract

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 immunopre-cipitation 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 tran-scription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.