Ameloblastin Endocytosis In Enamel Formation

Objectives: ABSTRACT:
Endocytosis is an essential function by ameloblasts to remove proteins during the course of enamel formation. Ameloblastin is an enamel protein secreted into the matrix and processed into peptides by enamel proteinases. The mechanisms or the pathways involved in endocytosis during enamel formation are not understood.
OBJECTIVE:
The goal of this study is to elucidate the endocytosis of ameloblastin (Ambn) during enamel formation in vivo and in vitro.
Methods: METHODS:
Wild-type mice were sacrificed at day 5 or 11. The hemi-mandibles were fixed, followed by decalcification and dehydration, then embedded in paraffin and sectioned. For mRNA localization, Rab 5, Rab7 and Ambn probes were hybridized in situ on day 5 and day 11 sectioned teeth. For immunolocalization tissue sections were incubated with anti-Rab5 and -Rab7 antibodies, and imaged under a light microscope. For in vitro experiments, Rab5 and Rab7 were localized with antibodies in LS8 cells. N-Ambn peptides were synthesized and labeled with Alexa-Fluor-488. For knock-down of Rab5, siRNAs was transduced into LS8 cells and N-Ambn-488 was added for 2 hrs at 5ug/mL. Cells were then fixed, and incubated with anti-Rab5 antibodies. Secondary antibody labeled with Alexa-Fluor-568 was used for fluorescence detection with laser confocal microscopy.
Results: RESULTS:
Rab5, Rab7 and Ambn mRNAs were detected in ameloblasts at secretory (Day 5) and maturation stages (Day 11) in mouse molars. Rab5 and Rab7 showed immunolocalization in the infranuclear region of both ameloblasts at secretory and maturation stages in mouse molars and LS8 cells. In LS8 cells, N-Ambn-488 co-localized with Rab5. Endocytosis of N-Ambn-488 was greatly reduced in the presence of Rab5 siRNA compared to control.
Conclusions: CONCLUSIONS:
mRNA and proteins involved in endocytosis (Rab5, Rab7, cathepsin D and cathepsin K) localized in ameloblasts. Ambn endocytosis associates with early endosomes.