TGFβ1-induced Amelotin gene expression is downregulated by Bax expression in mouse gingival epithelial cells

Amelotin (AMTN) is induced upon initiation of apoptosis by transforming growth factor beta1 (TGF beta 1) and is mediated by Smad3 in gingival epithelial cells (GE1 cells). This upregulation of AMTN gene expression is temporary, and the mechanism responsible is still unclear. The present study investigated the transcriptional downregulation of TGF beta 1-induced AMTN gene expression in GE1 cells during the progression of apoptosis. To examine time-dependent changes in the levels of AMTN, Smad3 and Bax mRNA induced by TGF beta 1, real-time PCR analyses were performed. Immunocytochemistry was carried out to detect the expression of Smad3 and Bax. Transient transfection analyses were performed using mouse AMTN gene promoter constructs of various lengths including Smad response elements (SBEs), in the presence or absence of TGF beta 1. Changes in Smad3 binding to SBEs resulting from overexpression of Bax were examined using ChIP assays. Overexpression of Bax dramatically downregulated the levels of TGF beta 1-induced AMTN mRNA and transcription of the AMTN gene. Smad3 binding to SBEs in the mouse AMTN gene promoter was induced by overexpression of Smad3 or TGF beta 1, and this was inhibited by Bax overexpression. These results show that the levels of AMTN mRNA induced by TGF beta 1 and Smad3 are decreased by robust expression of Bax in gingival epithelial cells.