Reuptake of Extracellular Amelogenin by Dental Epithelial Cells Results in Increased Levels of Amelogenin mRNA through Enhanced mRNA Stabilization

Abstract

Amelogenin is an extracellular matrix protein secreted by ameloblasts and is a major component of enamel matrix. Recently, in addition to their role in enamel formation, the biological activity of enamel proteins in the process of cell differentiation has recently become widely appreciated. In this study, we examined the biological activity of amelogenin on ameloblast differentiation. Recombinant mouse amelogenin (rm-amelogenin) enhanced the expression of endogenous amelogenin mRNA in a cultured dental epithelial cell line (HAT-7), despite a lack of increased amelogenin promoter activity. To solve this discrepancy, we analyzed the effects of rmamelogenin on the stability of amelogenin mRNA. The half-life of amelogenin mRNA is extremely short, but in the presence of rmamelogenin its half-life was extended three times longer than the control. Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells. It follows from our results that exogenous amelogenin increases amelogenin mRNA levels through stabilization of mRNA in the cytoplasm of HAT-7 cells. Here we speculated that during differentiation, dental epithelial cells utilize a unique mechanism for increasing the production of amelogenin, the reuptake of secreted amelogenin.

Critical role for αvβ6 integrin in enamel biomineralization

“…Amelogenin or its fragments are endocytosed by ameloblasts via a receptor-mediated mechanism involving LAMP1 and CD63 proteins (Shapiro et al, 2007). This uptake may serve as a feedback loop to upregulate amelogenin expression through stabilization of its mRNA in the cytoplasm (Xu et al, 2006). We did not find evidence for the direct involvement of avb6 integrin in amelogenin endocytosis.…”

Effect of BMP-2 on Gene Expression of Enamel Matrix Proteins at the Dental Epithelial Cell Line

“…The specific amelogenin gene splice products induce expression of the bone matrix proteins bone sialoprotein (BSP) and BAG-75 in culture and in an implant in vivo [5]. We previously demonstrated that the reuptake of full-length amelogenin protein results in increased levels of amelogenin mRNA through enhanced mRNA stabilization [6,7]. These findings indicated that amelogenin proteins are important for both ameloblast and odentoblast differentiation in an autocrine or paracrine manner.…”

“…The mRNA levels of differentiation-related marker genes were determined by quantitative real-time PCR as described previously [6,7,[16][17][18][19]. Briefly, total RNA was extracted at various points with ISOGEN (Nippon Gene, Japan).…”

“…HAT-7 cells, a dental epithelial cell line originating from the apical bud of a rat incisor [6,7,15], were cultured in Dulbecco's Modified Eagle's Medium (DMEM)/F-12 (GIBCO BRL, USA) supplemented with 10% fetal bovine serum and penicillin (100 units/ml)/streptomycin (100 μg/ml). All cultures were maintained in a humidified atmosphere of 5% CO 2 at 37°C.…”

Origin, Splicing, and Expression of Rodent Amelogenin Exon 8

“…For example, the Mmp20 null mouse and human kindreds with critical MMP20 mutations have brittle enamel that tends to delaminate from the dentin surface (Caterina et al, 2002;Kim et al, 2005;Ozdemir et al, 2005). It is believed that ameloblasts respond to changes in the extracellular matrix by adjusting their activities to maintain conditions appropriate for biomineralization (Xu et al, 2006).…”