CCAAT/enhancer-binding protein δ(C/EBPδ) maintains amelogenin expression in the absence of C/EBPα in vivo

Abstract

C/EBPα is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBPα-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBPα conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice bearing the floxed C/EBPα allele with keratin-14-Cre mice generate C/EBPα conditional knock-out mice. Real-time PCR analysis shows that removal of one C/EBPα allele from the molar enamel epithelial organ of 3-day postnatal mice results in dramatic decrease in endogenous C/EBPα mRNA levels and coordinately altered amelogenin mRNA abundance. Conditional deletion of both C/EBPα alleles further diminishes C/EBPα mRNA levels; however, rather than ablating amelogenin expression, we observe wild-type amelogenin mRNA abundance levels. We examined C/EBPβ and nuclear factor YA expression, two transcription factors that had previously been shown to modestly participate in amelogenin expression, in vitro but found no significant changes in either of their mRNA abundance levels comparing conditional knock-out mice with wild-type counterparts. Although the abundance of C/EBPδ is also unchanged in C/EBPα conditional knock-out mice, in vitro we find that C/EBPδ activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/EBPδ can functionally substitute for C/EBPα to produce an enamel matrix competent to direct biomineralization.